The document describes a proteomic comparison of Mediator complexes isolated from HeLa and SJSA-1 cells. Mass spectrometry analysis found that SJSA-1 cells have lower levels of several Mediator subunits compared to HeLa cells, and lack CDK8, CDK19, MED12 and other subunits that associate with the CDK8 module. The results support the hypothesis that unique factors are associated with Mediator in SJSA-1 cells, which have CDK19 but not CDK8.
SuperScript IV Reverse Transcriptase for RNA Analysis | ESHG 2015 Poster PS14...Thermo Fisher Scientific
Survey and interview studies conducted over a three year period revealed that researchers are not satisfied with their current reverse transcriptase and are performing reactions with increasingly difficult samples, such as poorly purified RNA and unpurified RNA (direct RT) that both contain inhibitors. To meet this performance gap, the Thermo Fisher Life Sciences Solutions group produced a new reverse transcriptase, SuperScript® IV, and experiments we performed show that it is the most robust reverse transcriptase compared to other enzymes. SuperScript® IV characterization was performed in the context of “real world” situations where users do not have perfect RNA samples. In the presence of a variety of inhibitors, we demonstrate that SuperScript® IV possesses superior performance in a variety of inhibitors, such as alcohols, salts, detergents, phenol, heparin, hematin, bile salts, and formalin typically found in sample preparation reagents, cell lines, blood, feces, and FFPE samples. This enzyme can even detect RNA targets in unpurified RNA samples (directly lysed cells) and whole blood without sacrificing sensitivity and yield. The introduction of SuperScript® IV enables researchers to obtain more consistent results independent of sample quality and simplify and speed up workflows by eliminating RNA purification.
DNA damage repair Neil3 gene Knockout in MOLT-4iosrjce
RNAi is superannuated cellular mechanism that protect organism against viruses that replicate
through double- stranded RNA. RNAi can be used to diminish gene expression from plasmid expressing and
inserted sequence repeat. A stable harpin would be expressed after the vector was integrated into the genome.
In this paper a shiRNA expressing vector for Neil3 was designed and developed which is capable of replication
in MOLT-4. This shiRNA vector had the ability to arose the RNAi pathway, and reduce the gene expression of
Neil3. This was assessed by using pSilence 4.1CMV as a vector, and Gapdh as positive control.
SuperScript IV Reverse Transcriptase for RNA Analysis | ESHG 2015 Poster PS14...Thermo Fisher Scientific
Survey and interview studies conducted over a three year period revealed that researchers are not satisfied with their current reverse transcriptase and are performing reactions with increasingly difficult samples, such as poorly purified RNA and unpurified RNA (direct RT) that both contain inhibitors. To meet this performance gap, the Thermo Fisher Life Sciences Solutions group produced a new reverse transcriptase, SuperScript® IV, and experiments we performed show that it is the most robust reverse transcriptase compared to other enzymes. SuperScript® IV characterization was performed in the context of “real world” situations where users do not have perfect RNA samples. In the presence of a variety of inhibitors, we demonstrate that SuperScript® IV possesses superior performance in a variety of inhibitors, such as alcohols, salts, detergents, phenol, heparin, hematin, bile salts, and formalin typically found in sample preparation reagents, cell lines, blood, feces, and FFPE samples. This enzyme can even detect RNA targets in unpurified RNA samples (directly lysed cells) and whole blood without sacrificing sensitivity and yield. The introduction of SuperScript® IV enables researchers to obtain more consistent results independent of sample quality and simplify and speed up workflows by eliminating RNA purification.
DNA damage repair Neil3 gene Knockout in MOLT-4iosrjce
RNAi is superannuated cellular mechanism that protect organism against viruses that replicate
through double- stranded RNA. RNAi can be used to diminish gene expression from plasmid expressing and
inserted sequence repeat. A stable harpin would be expressed after the vector was integrated into the genome.
In this paper a shiRNA expressing vector for Neil3 was designed and developed which is capable of replication
in MOLT-4. This shiRNA vector had the ability to arose the RNAi pathway, and reduce the gene expression of
Neil3. This was assessed by using pSilence 4.1CMV as a vector, and Gapdh as positive control.
Los días 20 y 21 de octubre de 2016, la Fundacion Ramón Areces organizó un simposio internacional para analizar las 'Enfermedades raras de la piel: de la clínica al gen y viceversa'. El doctor Fernando Larcher Laguzzi, del CIEMAT-Universidad Carlos III de Madrid-IIS Fundación Jiménez Díaz, ejerció de coordinador.
KDM5 epigenetic modifiers as a focus for drug discoveryChristopher Wynder
A summary presentation of my scientific work.
My laboratory focused on an enzyme KDM5b (aka PLU-1, JARID1b) that was widely expressed during development and played a key role in progression of breast cancer through HER-2.
My lab focused on understanding the key biochemical activity of the enzyme through dissecting the proteomic and genomic interactors.
Our results were confirmed through the use of ES cells, adult stem cells and mouse models.
Much of this work remains unpublished, please contact me for more information and/or access to any reagents that I still have as part of this work.
crwynder@gmail.com
WDR7 up-regulation upon knocking down of neighboring noncoding RNA using siRN...Vahid Erfani-Moghadam
Objective(s): Breast cancer is the second leading cause of cancer death in females. Understanding molecular mechanisms in cancer cells compared with normal cells is crucial for diagnostic and therapeutic strategies. Long intergenic non-protein coding RNA, a regulator of reprogramming (lincRNA-RoR) is a noncoding RNA which initially was detected in induced pluripotent stem cells, and it has an important role in cell reprogramming and highly expressed in breast cancer cells. A key point in successful gene silencing is the usage of siRNA delivery system that is safe and efficient. Materials and Methods: In this study, the fifth-generation of PAMAM dendrimer is used as a nanocarrier for entering siRNA molecules for gene silencing of lincRNA-RoR. WDR7 is the gene encoding adjacent of lincRNA-RoR, which has an important role in apoptosis and cell cycle. Gel retardation assay was used to find the best Negative/Positive (N/P) molar charge ratio of siRNA- PAMAM transfected into MDA-MB 231 cells. MTT assay was performed 24 hr after transfection revealed the IC50 value (half maximal inhibitory concentrations) about 100 nanomolar for lincRNA-ROR siRNA. Results: The lincRNA-RoR and WDR7 gene expression changes were evaluated by real-time PCR after siRNA treatment and showed an increase in the gene expression of WDR7. Conclusion: This study showed that PAMAM dendrimer G5/ siRNA could be a useful system delivery for future gene therapy approaches.
Nrf2 Transcription Factor- Nuclear Factor- Erythroid 2 related factor)PHARMA IQ EDUCATION
1. Nrf2- transcription factor
2. Reactive Oxygen Species
3. Free Radicals
4. Antioxidant Defence Mechanism
5. Function of Nrf2 receptor
6. Protein structural domain of Nrf2
7. Protein structural domain of Keap1
8. Physiological Role pf Nrf2
9.
THANK YOU
Los días 20 y 21 de octubre de 2016, la Fundacion Ramón Areces organizó un simposio internacional para analizar las 'Enfermedades raras de la piel: de la clínica al gen y viceversa'. El doctor Fernando Larcher Laguzzi, del CIEMAT-Universidad Carlos III de Madrid-IIS Fundación Jiménez Díaz, ejerció de coordinador.
KDM5 epigenetic modifiers as a focus for drug discoveryChristopher Wynder
A summary presentation of my scientific work.
My laboratory focused on an enzyme KDM5b (aka PLU-1, JARID1b) that was widely expressed during development and played a key role in progression of breast cancer through HER-2.
My lab focused on understanding the key biochemical activity of the enzyme through dissecting the proteomic and genomic interactors.
Our results were confirmed through the use of ES cells, adult stem cells and mouse models.
Much of this work remains unpublished, please contact me for more information and/or access to any reagents that I still have as part of this work.
crwynder@gmail.com
WDR7 up-regulation upon knocking down of neighboring noncoding RNA using siRN...Vahid Erfani-Moghadam
Objective(s): Breast cancer is the second leading cause of cancer death in females. Understanding molecular mechanisms in cancer cells compared with normal cells is crucial for diagnostic and therapeutic strategies. Long intergenic non-protein coding RNA, a regulator of reprogramming (lincRNA-RoR) is a noncoding RNA which initially was detected in induced pluripotent stem cells, and it has an important role in cell reprogramming and highly expressed in breast cancer cells. A key point in successful gene silencing is the usage of siRNA delivery system that is safe and efficient. Materials and Methods: In this study, the fifth-generation of PAMAM dendrimer is used as a nanocarrier for entering siRNA molecules for gene silencing of lincRNA-RoR. WDR7 is the gene encoding adjacent of lincRNA-RoR, which has an important role in apoptosis and cell cycle. Gel retardation assay was used to find the best Negative/Positive (N/P) molar charge ratio of siRNA- PAMAM transfected into MDA-MB 231 cells. MTT assay was performed 24 hr after transfection revealed the IC50 value (half maximal inhibitory concentrations) about 100 nanomolar for lincRNA-ROR siRNA. Results: The lincRNA-RoR and WDR7 gene expression changes were evaluated by real-time PCR after siRNA treatment and showed an increase in the gene expression of WDR7. Conclusion: This study showed that PAMAM dendrimer G5/ siRNA could be a useful system delivery for future gene therapy approaches.
Nrf2 Transcription Factor- Nuclear Factor- Erythroid 2 related factor)PHARMA IQ EDUCATION
1. Nrf2- transcription factor
2. Reactive Oxygen Species
3. Free Radicals
4. Antioxidant Defence Mechanism
5. Function of Nrf2 receptor
6. Protein structural domain of Nrf2
7. Protein structural domain of Keap1
8. Physiological Role pf Nrf2
9.
THANK YOU
Each technological age has been marked by a shift in how the industrial platform enables companies to rethink their business processes and create wealth. In the talk I argue that we are limiting our view of what this next industrial/digital age can offer because of how we read, measure and through that perceive the world (how we cherry pick data). Companies are locked in metrics and quantitative measures, data that can fit into a spreadsheet. And by that they see the digital transformation merely as an efficiency tool to the fossil fuel age. But we need to stretch further…
Covid-19, commonly known as Coronavirus, is a single-stranded positive-sense RNA virus. It is a known fact that RNA-duplex and RNA-DNA duplex is thermodynamically more stable than ds-DNA which in turn is more stable than ss-RNA i.e. it requires more harsh conditions (Like higher temperature) to denature ds-RNA than ds-DNA. So, injecting a modified anti-sense RNA would effectively arrest RNA proliferation by forming a near-neutral duplex (i.e. this Duplex can't be proofread stopping the retrosynthesis) in a Corona-affected patient, which is the key idea of my project.
Bioinformática y supercomputación. Razones para hacerse bioinformático en la UMAM. Gonzalo Claros
¿En qué consiste la bioinformática? ¿Cómo puedo especializarme? ¿Dónde? Capacidad de supercomputación en la UMA. Recientes logros bioinformáticos relacionados con la medicina y con la ciencia en general, muchos de ellos realizados por equipos de la UMA.
Aptamers provide opportunities for structure-based drug design strategies relevant to therapeutic intervention. Recent advances in the chemical modifications of nucleic acids suggest that one of the major barriers to use, stability, can be overcome. The high affinity and specificity of aptamers rival antibodies and make them a promising tool in diagnostic and therapeutic application. We should expect more aptamers to be isolated in the near future against an ever increasing repertoire of targets, using these different SELEX approaches with increased speed and efficiency. Aptamers are poised to successfully compete with monoclonal Abs in therapeutics and drug development within the next few decades.
Optimization of experimental protocols for cellular lysisExpedeon
In this project, we have compared existing sample preparation methods for proteomics studies against newly developed FASP method and our in-house developed SDS-TCA protocol. For our
preliminary studies, we have chosen a very well characterized soil microbe Pseudomonas putida.
Nucleic Acids (DNA/RNA) as Nanoparticles Structures for siRNA Delivery Medica...Al Baha University
Engineered design of synthetic DNA/RNA molecules can generate pre-defined structures that can easily self-assemble to form nanoparticles with multiple functionalities. The identification and selection of highly potent siRNA sequences has already been accomplished for many gene targets, and the synthesis of siRNAs on a large scale has been achieved. The field of oligonucleotide-based nanotechnology for biomedical applications is just emerging, but will play an important role in the delivery of siRNA. In particular, oligonucleotide-based structural RNAi systems described in this chapter are promising as a new generation of gene delivery carriers for cancer therapy.
RNA interference (RNAi) is a gene regulation mechanism initiated by RNA molecules that enables sequence-specific gene silencing by promoting degradation of specific mRNAs. Molecular therapy using small interfering RNA (siRNA) has shown great therapeutic potential for diseases caused by abnormal gene overexpression or mutation. The major challenges to application of siRNA therapeutics include the stability and effective delivery of siRNA in vivo. In this chapter, we discuss recent advances in nanoparticle-mediated siRNA delivery systems and the application of these systems in clinical trials for cancer therapy. Furthermore, we offer perspectives on future applications of siRNA therapeutics.
1. A proteomic comparison of Mediator in HeLa
and SJSA cells
Will Daggett
Group Meeting
April 13th, 2015
2. CDK8 / CDK19 associate with mediator through
a four-protein module
CDK8 module
MED13/LMED12/L
cyclin C
CDK8/19
wt
CDK8 module CDK8 Mediator
Mediator
3. SJSA-1 cells have CDK19, but not CDK8 by western blot
CDK8 and 19 are very similar – hard to study in isolation
SJSA-1 (osteosarcoma) cells are easy to work with
Galbraith et al. Cell. 2013.
4. 1. Do SJSA-1 cells truly have CDK19, but not CDK8? How about
other differences in Mediator subunits?
2. Are there unique factors associated with CDK19-Mediator
in SJSAs?
Questions
5. Hypotheses
1. Unique factors are associated with Mediator in SJSAs
2. SJSA-1 cells have less CDK8, but do have CDK19
6. Approach
1. IP MED1 from SJSA (CDK19) and HeLa (CDK8/19) NE’s
a. Submit samples for mass spectrometry
b. Compare Mediator subunits in both cell lines
c. Other unique factors?
2. IP CDK19 from SJSAs and HeLa
a. Evaluate CDK19 IP efficiency – CDK8/19 antibodies
b. Compare CDK19-Mediator associated proteins
7. Silver Stain HeLa NE and CDK8 antibody
250 kD
150
100
75
50
Silver Stain HeLa NE and CDK8 antibody
250 kD
150
100
75
50
37
37
Antibody amount optimization and IP practice – CDK8
8. Antibody amount optimization and IP practice – MED1
250 kD
150
100
75
50
25
37
Silver Stain HeLa NE and MED1 antibody
9. MED1 IPs submitted for MS analysis – total protein
Sypro Ruby
10
BSA (ng)
20 50 100 150
250
150
100
75
50
37
25
For IP:
-20ug MED1 antibody on 20ul dry protein G beads
-Equivalent amounts of total protein in NE (10mg/ml HeLa; 2mg/ml SJSA)
-2.5 bead volume elutions in low pH glycine
***Found about a 4x difference between the samples***
10. MED1 IPs submitted for MS analysis - Mediator
250
150
100
75
50
37
25
MED12 (1:1k)
MED23 (1:1k)
CDK8 (1:1k)
Cyc C (1:1k)
HeLa E1+2
1, 2, 5%
HeLa E3
1, 2%
SJSA E1+2
1, 2, 5%
SJSA E3
1, 2%
SJSA IP has less total protein
Submit all of SJSA IP for MS – equivalent amount of HeLa/beads
SJSA IP has less Mediator
SJSA IP has no CDK8 and MED12!
Western
***8x difference according to MED23 – used 5x less HeLa eluate***
12. MS standardization with spectral counts - NSAF
Allows us to directly compare proteins across samples!
Must standardize for total spectral counts per sample
Zhang et al. Anal. Chem. 2010.
Where, SAF (spectral abundance factor) = prot. spec. cts / length
i = protein identity
N = total number of proteins
Must standardize for protein length
13. Normalized Mediator comparison with IP-MS – NSAF x 10^3
Gene Description beads HeLa SJSA (HeLa/SJSA) (SJSA/HeLa)
MED1_HUMAN Mediator of RNA polymerase II transcription subunit 1 0 5.0 4.7 1.07 0.93
MED4_HUMAN Mediator of RNA polymerase II transcription subunit 4 0 5.9 8.2 0.71 1.40
MED6_HUMAN Mediator of RNA polymerase II transcription subunit 6 0 5.7 7.6 0.76 1.32
MED7_HUMAN Mediator of RNA polymerase II transcription subunit 7 0 3.1 7.6 0.40 2.49
MED8_HUMAN Mediator of RNA polymerase II transcription subunit 8 0 3.5 13.3 0.26 3.80
MED9_HUMAN Mediator of RNA polymerase II transcription subunit 9 0 2.2 2.7 0.82 1.22
MED10_HUMAN Mediator of RNA polymerase II transcription subunit 10 0 6.4 11.1 0.58 1.73
MED11_HUMAN Mediator of RNA polymerase II transcription subunit 11 0 1.0 1.0 1.00 1.00
MED14_HUMAN Mediator of RNA polymerase II transcription subunit 14 0 3.5 7.2 0.48 2.07
MED15_HUMAN Isoform 3 of Mediator of RNA polymerase II transcription subunit 15 0 4.1 4.9 0.84 1.19
MED16_HUMAN Isoform 2 of Mediator of RNA polymerase II transcription subunit 16 0 2.7 7.2 0.38 2.64
MED17_HUMAN Mediator of RNA polymerase II transcription subunit 17 0 4.8 8.8 0.54 1.84
MED18_HUMAN Mediator of RNA polymerase II transcription subunit 18 0 2.6 1.7 1.53 0.65
MED19_HUMAN Mediator of RNA polymerase II transcription subunit 19 0 1.9 1.3 1.45 0.69
MED20_HUMAN Mediator of RNA polymerase II transcription subunit 20 0 6.8 7.7 0.89 1.12
MED21_HUMAN Mediator of RNA polymerase II transcription subunit 21 0 2.5 8.2 0.30 3.32
MED22_HUMAN Isoform Surf5A of Mediator of RNA polymerase II transcription subunit 22 0 4.0 9.0 0.44 2.25
MED23_HUMAN Isoform 4 of Mediator of RNA polymerase II transcription subunit 23 0 1.6 5.8 0.27 3.69
MED24_HUMAN Isoform 2 of Mediator of RNA polymerase II transcription subunit 24 0 2.8 9.8 0.28 3.52
MED25_HUMAN Isoform 5 of Mediator of RNA polymerase II transcription subunit 25 0 0.8 2.1 0.41 2.45
MED26_HUMAN Mediator of RNA polymerase II transcription subunit 26 0 1.8 0 X X
MED27_HUMAN Mediator of RNA polymerase II transcription subunit 27 0 2.2 15.3 0.15 6.81
MED28_HUMAN Mediator of RNA polymerase II transcription subunit 28 0 1.2 1.8 0.70 1.43
MED29_HUMAN Mediator of RNA polymerase II transcription subunit 29 0 3.9 5.7 0.68 1.47
MED30_HUMAN Mediator of RNA polymerase II transcription subunit 30 0 3.9 17.3 0.23 4.42
MED31_HUMAN Mediator of RNA polymerase II transcription subunit 31 0 4.4 4.5 0.97 1.03
CDK8_HUMAN Isoform 2 of Cyclin-dependent kinase 8 0 1.3 0 X X
CDK19_HUMAN Cyclin-dependent kinase 19 0 1.1 0 X X
CCNC_HUMAN Isoform 2 of Cyclin-C 0 2.1 0 X X
MED12_HUMAN Isoform 3 of Mediator of RNA polymerase II transcription subunit 12 0 2.3 0 X X
MED13_HUMAN Mediator of RNA polymerase II transcription subunit 13 0 1.2 1.2 0.99 1.01
MD13L_HUMAN Mediator of RNA polymerase II transcription subunit 13-like 0 0.2 0.3 0.65 1.55
Sum of SAFs per sample: Beads (HeLa) = 45.7 HeLa MED1 = 29.4 SJSA MED1 = 12.6
14. Normalized Mediator comparison with IP-MS – NSAF x 10^3
Gene Description beads HeLa SJSA (HeLa/SJSA) (SJSA/HeLa)
MED1_HUMAN Mediator of RNA polymerase II transcription subunit 1 0 5.0 4.7 1.07 0.93
MED4_HUMAN Mediator of RNA polymerase II transcription subunit 4 0 5.9 8.2 0.71 1.40
MED6_HUMAN Mediator of RNA polymerase II transcription subunit 6 0 5.7 7.6 0.76 1.32
MED7_HUMAN Mediator of RNA polymerase II transcription subunit 7 0 3.1 7.6 0.40 2.49
MED8_HUMAN Mediator of RNA polymerase II transcription subunit 8 0 3.5 13.3 0.26 3.80
MED9_HUMAN Mediator of RNA polymerase II transcription subunit 9 0 2.2 2.7 0.82 1.22
MED10_HUMAN Mediator of RNA polymerase II transcription subunit 10 0 6.4 11.1 0.58 1.73
MED11_HUMAN Mediator of RNA polymerase II transcription subunit 11 0 1.0 1.0 1.00 1.00
MED14_HUMAN Mediator of RNA polymerase II transcription subunit 14 0 3.5 7.2 0.48 2.07
MED15_HUMAN Isoform 3 of Mediator of RNA polymerase II transcription subunit 15 0 4.1 4.9 0.84 1.19
MED16_HUMAN Isoform 2 of Mediator of RNA polymerase II transcription subunit 16 0 2.7 7.2 0.38 2.64
MED17_HUMAN Mediator of RNA polymerase II transcription subunit 17 0 4.8 8.8 0.54 1.84
MED18_HUMAN Mediator of RNA polymerase II transcription subunit 18 0 2.6 1.7 1.53 0.65
MED19_HUMAN Mediator of RNA polymerase II transcription subunit 19 0 1.9 1.3 1.45 0.69
MED20_HUMAN Mediator of RNA polymerase II transcription subunit 20 0 6.8 7.7 0.89 1.12
MED21_HUMAN Mediator of RNA polymerase II transcription subunit 21 0 2.5 8.2 0.30 3.32
MED22_HUMAN Isoform Surf5A of Mediator of RNA polymerase II transcription subunit 22 0 4.0 9.0 0.44 2.25
MED23_HUMAN Isoform 4 of Mediator of RNA polymerase II transcription subunit 23 0 1.6 5.8 0.27 3.69
MED24_HUMAN Isoform 2 of Mediator of RNA polymerase II transcription subunit 24 0 2.8 9.8 0.28 3.52
MED25_HUMAN Isoform 5 of Mediator of RNA polymerase II transcription subunit 25 0 0.8 2.1 0.41 2.45
MED26_HUMAN Mediator of RNA polymerase II transcription subunit 26 0 1.8 0 X X
MED27_HUMAN Mediator of RNA polymerase II transcription subunit 27 0 2.2 15.3 0.15 6.81
MED28_HUMAN Mediator of RNA polymerase II transcription subunit 28 0 1.2 1.8 0.70 1.43
MED29_HUMAN Mediator of RNA polymerase II transcription subunit 29 0 3.9 5.7 0.68 1.47
MED30_HUMAN Mediator of RNA polymerase II transcription subunit 30 0 3.9 17.3 0.23 4.42
MED31_HUMAN Mediator of RNA polymerase II transcription subunit 31 0 4.4 4.5 0.97 1.03
CDK8_HUMAN Isoform 2 of Cyclin-dependent kinase 8 0 1.3 0 X X
CDK19_HUMAN Cyclin-dependent kinase 19 0 1.1 0 X X
CCNC_HUMAN Isoform 2 of Cyclin-C 0 2.1 0 X X
MED12_HUMAN Isoform 3 of Mediator of RNA polymerase II transcription subunit 12 0 2.3 0 X X
MED13_HUMAN Mediator of RNA polymerase II transcription subunit 13 0 1.2 1.2 0.99 1.01
MD13L_HUMAN Mediator of RNA polymerase II transcription subunit 13-like 0 0.2 0.3 0.65 1.55
Sum of SAFs per sample: Beads (HeLa) = 45.7 HeLa MED1 = 29.4 SJSA MED1 = 12.6
15. Normalized Mediator comparison with IP-MS – NSAF x 10^3
Gene Description beads HeLa SJSA (HeLa/SJSA) (SJSA/HeLa)
MED1_HUMAN Mediator of RNA polymerase II transcription subunit 1 0 5.0 4.7 1.07 0.93
MED4_HUMAN Mediator of RNA polymerase II transcription subunit 4 0 5.9 8.2 0.71 1.40
MED6_HUMAN Mediator of RNA polymerase II transcription subunit 6 0 5.7 7.6 0.76 1.32
MED7_HUMAN Mediator of RNA polymerase II transcription subunit 7 0 3.1 7.6 0.40 2.49
MED8_HUMAN Mediator of RNA polymerase II transcription subunit 8 0 3.5 13.3 0.26 3.80
MED9_HUMAN Mediator of RNA polymerase II transcription subunit 9 0 2.2 2.7 0.82 1.22
MED10_HUMAN Mediator of RNA polymerase II transcription subunit 10 0 6.4 11.1 0.58 1.73
MED11_HUMAN Mediator of RNA polymerase II transcription subunit 11 0 1.0 1.0 1.00 1.00
MED14_HUMAN Mediator of RNA polymerase II transcription subunit 14 0 3.5 7.2 0.48 2.07
MED15_HUMAN Isoform 3 of Mediator of RNA polymerase II transcription subunit 15 0 4.1 4.9 0.84 1.19
MED16_HUMAN Isoform 2 of Mediator of RNA polymerase II transcription subunit 16 0 2.7 7.2 0.38 2.64
MED17_HUMAN Mediator of RNA polymerase II transcription subunit 17 0 4.8 8.8 0.54 1.84
MED18_HUMAN Mediator of RNA polymerase II transcription subunit 18 0 2.6 1.7 1.53 0.65
MED19_HUMAN Mediator of RNA polymerase II transcription subunit 19 0 1.9 1.3 1.45 0.69
MED20_HUMAN Mediator of RNA polymerase II transcription subunit 20 0 6.8 7.7 0.89 1.12
MED21_HUMAN Mediator of RNA polymerase II transcription subunit 21 0 2.5 8.2 0.30 3.32
MED22_HUMAN Isoform Surf5A of Mediator of RNA polymerase II transcription subunit 22 0 4.0 9.0 0.44 2.25
MED23_HUMAN Isoform 4 of Mediator of RNA polymerase II transcription subunit 23 0 1.6 5.8 0.27 3.69
MED24_HUMAN Isoform 2 of Mediator of RNA polymerase II transcription subunit 24 0 2.8 9.8 0.28 3.52
MED25_HUMAN Isoform 5 of Mediator of RNA polymerase II transcription subunit 25 0 0.8 2.1 0.41 2.45
MED26_HUMAN Mediator of RNA polymerase II transcription subunit 26 0 1.8 0 X X
MED27_HUMAN Mediator of RNA polymerase II transcription subunit 27 0 2.2 15.3 0.15 6.81
MED28_HUMAN Mediator of RNA polymerase II transcription subunit 28 0 1.2 1.8 0.70 1.43
MED29_HUMAN Mediator of RNA polymerase II transcription subunit 29 0 3.9 5.7 0.68 1.47
MED30_HUMAN Mediator of RNA polymerase II transcription subunit 30 0 3.9 17.3 0.23 4.42
MED31_HUMAN Mediator of RNA polymerase II transcription subunit 31 0 4.4 4.5 0.97 1.03
CDK8_HUMAN Isoform 2 of Cyclin-dependent kinase 8 0 1.3 0 X X
CDK19_HUMAN Cyclin-dependent kinase 19 0 1.1 0 X X
CCNC_HUMAN Isoform 2 of Cyclin-C 0 2.1 0 X X
MED12_HUMAN Isoform 3 of Mediator of RNA polymerase II transcription subunit 12 0 2.3 0 X X
MED13_HUMAN Mediator of RNA polymerase II transcription subunit 13 0 1.2 1.2 0.99 1.01
MD13L_HUMAN Mediator of RNA polymerase II transcription subunit 13-like 0 0.2 0.3 0.65 1.55
Sum of SAFs per sample: Beads (HeLa) = 45.7 HeLa MED1 = 29.4 SJSA MED1 = 12.6
16. Normalized Mediator comparison with IP-MS – NSAF x 10^3
Gene Description beads HeLa SJSA (HeLa/SJSA) (SJSA/HeLa)
MED1_HUMAN Mediator of RNA polymerase II transcription subunit 1 0 5.0 4.7 1.07 0.93
MED4_HUMAN Mediator of RNA polymerase II transcription subunit 4 0 5.9 8.2 0.71 1.40
MED6_HUMAN Mediator of RNA polymerase II transcription subunit 6 0 5.7 7.6 0.76 1.32
MED7_HUMAN Mediator of RNA polymerase II transcription subunit 7 0 3.1 7.6 0.40 2.49
MED8_HUMAN Mediator of RNA polymerase II transcription subunit 8 0 3.5 13.3 0.26 3.80
MED9_HUMAN Mediator of RNA polymerase II transcription subunit 9 0 2.2 2.7 0.82 1.22
MED10_HUMAN Mediator of RNA polymerase II transcription subunit 10 0 6.4 11.1 0.58 1.73
MED11_HUMAN Mediator of RNA polymerase II transcription subunit 11 0 1.0 1.0 1.00 1.00
MED14_HUMAN Mediator of RNA polymerase II transcription subunit 14 0 3.5 7.2 0.48 2.07
MED15_HUMAN Isoform 3 of Mediator of RNA polymerase II transcription subunit 15 0 4.1 4.9 0.84 1.19
MED16_HUMAN Isoform 2 of Mediator of RNA polymerase II transcription subunit 16 0 2.7 7.2 0.38 2.64
MED17_HUMAN Mediator of RNA polymerase II transcription subunit 17 0 4.8 8.8 0.54 1.84
MED18_HUMAN Mediator of RNA polymerase II transcription subunit 18 0 2.6 1.7 1.53 0.65
MED19_HUMAN Mediator of RNA polymerase II transcription subunit 19 0 1.9 1.3 1.45 0.69
MED20_HUMAN Mediator of RNA polymerase II transcription subunit 20 0 6.8 7.7 0.89 1.12
MED21_HUMAN Mediator of RNA polymerase II transcription subunit 21 0 2.5 8.2 0.30 3.32
MED22_HUMAN Isoform Surf5A of Mediator of RNA polymerase II transcription subunit 22 0 4.0 9.0 0.44 2.25
MED23_HUMAN Isoform 4 of Mediator of RNA polymerase II transcription subunit 23 0 1.6 5.8 0.27 3.69
MED24_HUMAN Isoform 2 of Mediator of RNA polymerase II transcription subunit 24 0 2.8 9.8 0.28 3.52
MED25_HUMAN Isoform 5 of Mediator of RNA polymerase II transcription subunit 25 0 0.8 2.1 0.41 2.45
MED26_HUMAN Mediator of RNA polymerase II transcription subunit 26 0 1.8 0 X X
MED27_HUMAN Mediator of RNA polymerase II transcription subunit 27 0 2.2 15.3 0.15 6.81
MED28_HUMAN Mediator of RNA polymerase II transcription subunit 28 0 1.2 1.8 0.70 1.43
MED29_HUMAN Mediator of RNA polymerase II transcription subunit 29 0 3.9 5.7 0.68 1.47
MED30_HUMAN Mediator of RNA polymerase II transcription subunit 30 0 3.9 17.3 0.23 4.42
MED31_HUMAN Mediator of RNA polymerase II transcription subunit 31 0 4.4 4.5 0.97 1.03
CDK8_HUMAN Isoform 2 of Cyclin-dependent kinase 8 0 1.3 0 X X
CDK19_HUMAN Cyclin-dependent kinase 19 0 1.1 0 X X
CCNC_HUMAN Isoform 2 of Cyclin-C 0 2.1 0 X X
MED12_HUMAN Isoform 3 of Mediator of RNA polymerase II transcription subunit 12 0 2.3 0 X X
MED13_HUMAN Mediator of RNA polymerase II transcription subunit 13 0 1.2 1.2 0.99 1.01
MD13L_HUMAN Mediator of RNA polymerase II transcription subunit 13-like 0 0.2 0.3 0.65 1.55
Sum of SAFs per sample: Beads (HeLa) = 45.7 HeLa MED1 = 29.4 SJSA MED1 = 12.6
17. Normalized Mediator comparison with IP-MS – NSAF x 10^3
Gene Description beads HeLa SJSA (HeLa/SJSA) (SJSA/HeLa)
MED1_HUMAN Mediator of RNA polymerase II transcription subunit 1 0 5.0 4.7 1.07 0.93
MED4_HUMAN Mediator of RNA polymerase II transcription subunit 4 0 5.9 8.2 0.71 1.40
MED6_HUMAN Mediator of RNA polymerase II transcription subunit 6 0 5.7 7.6 0.76 1.32
MED7_HUMAN Mediator of RNA polymerase II transcription subunit 7 0 3.1 7.6 0.40 2.49
MED8_HUMAN Mediator of RNA polymerase II transcription subunit 8 0 3.5 13.3 0.26 3.80
MED9_HUMAN Mediator of RNA polymerase II transcription subunit 9 0 2.2 2.7 0.82 1.22
MED10_HUMAN Mediator of RNA polymerase II transcription subunit 10 0 6.4 11.1 0.58 1.73
MED11_HUMAN Mediator of RNA polymerase II transcription subunit 11 0 1.0 1.0 1.00 1.00
MED14_HUMAN Mediator of RNA polymerase II transcription subunit 14 0 3.5 7.2 0.48 2.07
MED15_HUMAN Isoform 3 of Mediator of RNA polymerase II transcription subunit 15 0 4.1 4.9 0.84 1.19
MED16_HUMAN Isoform 2 of Mediator of RNA polymerase II transcription subunit 16 0 2.7 7.2 0.38 2.64
MED17_HUMAN Mediator of RNA polymerase II transcription subunit 17 0 4.8 8.8 0.54 1.84
MED18_HUMAN Mediator of RNA polymerase II transcription subunit 18 0 2.6 1.7 1.53 0.65
MED19_HUMAN Mediator of RNA polymerase II transcription subunit 19 0 1.9 1.3 1.45 0.69
MED20_HUMAN Mediator of RNA polymerase II transcription subunit 20 0 6.8 7.7 0.89 1.12
MED21_HUMAN Mediator of RNA polymerase II transcription subunit 21 0 2.5 8.2 0.30 3.32
MED22_HUMAN Isoform Surf5A of Mediator of RNA polymerase II transcription subunit 22 0 4.0 9.0 0.44 2.25
MED23_HUMAN Isoform 4 of Mediator of RNA polymerase II transcription subunit 23 0 1.6 5.8 0.27 3.69
MED24_HUMAN Isoform 2 of Mediator of RNA polymerase II transcription subunit 24 0 2.8 9.8 0.28 3.52
MED25_HUMAN Isoform 5 of Mediator of RNA polymerase II transcription subunit 25 0 0.8 2.1 0.41 2.45
MED26_HUMAN Mediator of RNA polymerase II transcription subunit 26 0 1.8 0 X X
MED27_HUMAN Mediator of RNA polymerase II transcription subunit 27 0 2.2 15.3 0.15 6.81
MED28_HUMAN Mediator of RNA polymerase II transcription subunit 28 0 1.2 1.8 0.70 1.43
MED29_HUMAN Mediator of RNA polymerase II transcription subunit 29 0 3.9 5.7 0.68 1.47
MED30_HUMAN Mediator of RNA polymerase II transcription subunit 30 0 3.9 17.3 0.23 4.42
MED31_HUMAN Mediator of RNA polymerase II transcription subunit 31 0 4.4 4.5 0.97 1.03
CDK8_HUMAN Isoform 2 of Cyclin-dependent kinase 8 0 1.3 0 X X
CDK19_HUMAN Cyclin-dependent kinase 19 0 1.1 0 X X
CCNC_HUMAN Isoform 2 of Cyclin-C 0 2.1 0 X X
MED12_HUMAN Isoform 3 of Mediator of RNA polymerase II transcription subunit 12 0 2.3 0 X X
MED13_HUMAN Mediator of RNA polymerase II transcription subunit 13 0 1.2 1.2 0.99 1.01
MD13L_HUMAN Mediator of RNA polymerase II transcription subunit 13-like 0 0.2 0.3 0.65 1.55
Sum of SAFs per sample: Beads (HeLa) = 45.7 HeLa MED1 = 29.4 SJSA MED1 = 12.6
?
18. Normalized Mediator comparison with IP-MS – NSAF x 10^3
Gene Description beads HeLa SJSA (HeLa/SJSA) (SJSA/HeLa)
MED1_HUMAN Mediator of RNA polymerase II transcription subunit 1 0 5.0 4.7 1.07 0.93
MED4_HUMAN Mediator of RNA polymerase II transcription subunit 4 0 5.9 8.2 0.71 1.40
MED6_HUMAN Mediator of RNA polymerase II transcription subunit 6 0 5.7 7.6 0.76 1.32
MED7_HUMAN Mediator of RNA polymerase II transcription subunit 7 0 3.1 7.6 0.40 2.49
MED8_HUMAN Mediator of RNA polymerase II transcription subunit 8 0 3.5 13.3 0.26 3.80
MED9_HUMAN Mediator of RNA polymerase II transcription subunit 9 0 2.2 2.7 0.82 1.22
MED10_HUMAN Mediator of RNA polymerase II transcription subunit 10 0 6.4 11.1 0.58 1.73
MED11_HUMAN Mediator of RNA polymerase II transcription subunit 11 0 1.0 1.0 1.00 1.00
MED14_HUMAN Mediator of RNA polymerase II transcription subunit 14 0 3.5 7.2 0.48 2.07
MED15_HUMAN Isoform 3 of Mediator of RNA polymerase II transcription subunit 15 0 4.1 4.9 0.84 1.19
MED16_HUMAN Isoform 2 of Mediator of RNA polymerase II transcription subunit 16 0 2.7 7.2 0.38 2.64
MED17_HUMAN Mediator of RNA polymerase II transcription subunit 17 0 4.8 8.8 0.54 1.84
MED18_HUMAN Mediator of RNA polymerase II transcription subunit 18 0 2.6 1.7 1.53 0.65
MED19_HUMAN Mediator of RNA polymerase II transcription subunit 19 0 1.9 1.3 1.45 0.69
MED20_HUMAN Mediator of RNA polymerase II transcription subunit 20 0 6.8 7.7 0.89 1.12
MED21_HUMAN Mediator of RNA polymerase II transcription subunit 21 0 2.5 8.2 0.30 3.32
MED22_HUMAN Isoform Surf5A of Mediator of RNA polymerase II transcription subunit 22 0 4.0 9.0 0.44 2.25
MED23_HUMAN Isoform 4 of Mediator of RNA polymerase II transcription subunit 23 0 1.6 5.8 0.27 3.69
MED24_HUMAN Isoform 2 of Mediator of RNA polymerase II transcription subunit 24 0 2.8 9.8 0.28 3.52
MED25_HUMAN Isoform 5 of Mediator of RNA polymerase II transcription subunit 25 0 0.8 2.1 0.41 2.45
MED26_HUMAN Mediator of RNA polymerase II transcription subunit 26 0 1.8 0 X X
MED27_HUMAN Mediator of RNA polymerase II transcription subunit 27 0 2.2 15.3 0.15 6.81
MED28_HUMAN Mediator of RNA polymerase II transcription subunit 28 0 1.2 1.8 0.70 1.43
MED29_HUMAN Mediator of RNA polymerase II transcription subunit 29 0 3.9 5.7 0.68 1.47
MED30_HUMAN Mediator of RNA polymerase II transcription subunit 30 0 3.9 17.3 0.23 4.42
MED31_HUMAN Mediator of RNA polymerase II transcription subunit 31 0 4.4 4.5 0.97 1.03
CDK8_HUMAN Isoform 2 of Cyclin-dependent kinase 8 0 1.3 0 X X
CDK19_HUMAN Cyclin-dependent kinase 19 0 1.1 0 X X
CCNC_HUMAN Isoform 2 of Cyclin-C 0 2.1 0 X X
MED12_HUMAN Isoform 3 of Mediator of RNA polymerase II transcription subunit 12 0 2.3 0 X X
MED13_HUMAN Mediator of RNA polymerase II transcription subunit 13 0 1.2 1.2 0.99 1.01
MD13L_HUMAN Mediator of RNA polymerase II transcription subunit 13-like 0 0.2 0.3 0.65 1.55
Sum of SAFs per sample: Beads (HeLa) = 45.7 HeLa MED1 = 29.4 SJSA MED1 = 12.6
?
19. Mediator abundance with MaxQuant
CDK19/CCNC are likely limit of detection issues! Few peptides ID’d in HeLa
MED12 and MED26 are likely NOT limit of detection issues – not there in SJSAs!
Gene names Protein names Un. peps-beads Intensity-beads Un. peps-HeLa Intensity-HeLa Un. peps-SJSA Intensity-SJSA HeLa/SJSA SJSA/HeLa
MED1 Mediator of RNA polymerase II transcription subunit 1 0 0 36 1137800000 19 761510000 1.49 0.67
MED4 Mediator of RNA polymerase II transcription subunit 4 0 0 8 281240000 7 376590000 0.75 1.34
MED6 Mediator of RNA polymerase II transcription subunit 6 0 0 4 135590000 4 247150000 0.55 1.82
MED7 Mediator of RNA polymerase II transcription subunit 7 0 0 4 35440000 3 61751000 0.57 1.74
MED8 Mediator of RNA polymerase II transcription subunit 8 0 0 6 131310000 7 473170000 0.28 3.60
MED9 Mediator of RNA polymerase II transcription subunit 9 0 0 3 58757000 2 33613000 1.75 0.57
MED10 Mediator of RNA polymerase II transcription subunit 10 0 0 1 14584000 3 89406000 0.16 6.13
MED11 Mediator of RNA polymerase II transcription subunit 11 0 0 1 11409000 1 5959700 1.91 0.52
MED14 Mediator of RNA polymerase II transcription subunit 14 0 0 18 325090000 30 1106300000 0.29 3.40
MED15 Mediator of RNA polymerase II transcription subunit 15 0 0 10 210060000 6 308430000 0.68 1.47
MED16 Mediator of RNA polymerase II transcription subunit 16 0 0 12 466610000 14 743740000 0.63 1.59
MED17 Mediator of RNA polymerase II transcription subunit 17 0 0 13 280930000 15 528550000 0.53 1.88
MED18 Mediator of RNA polymerase II transcription subunit 18 0 0 5 111340000 2 66021000 1.69 0.59
MED19 Mediator of RNA polymerase II transcription subunit 19 0 0 3 62846000 2 27582000 2.28 0.44
MED20 Mediator of RNA polymerase II transcription subunit 20 0 0 6 216990000 6 307540000 0.71 1.42
MED21 Mediator of RNA polymerase II transcription subunit 21 0 0 1 4579300 2 296360000 0.02 64.72
MED22 Mediator of RNA polymerase II transcription subunit 22 0 0 4 90392000 3 102540000 0.88 1.13
MED23 Mediator of RNA polymerase II transcription subunit 23 0 0 13 142590000 19 543390000 0.26 3.81
MED24 Mediator of RNA polymerase II transcription subunit 24 0 0 13 234470000 19 672050000 0.35 2.87
MED25 Mediator of RNA polymerase II transcription subunit 25 0 0 4 19285000 2 54583000 0.35 2.83
MED26 Mediator of RNA polymerase II transcription subunit 26 0 0 9 88457000 0 0 X 0
MED27 Mediator of RNA polymerase II transcription subunit 27 0 0 5 139100000 9 400350000 0.35 2.88
MED28 Mediator of RNA polymerase II transcription subunit 28 0 0 1 8762400 1 14993000 0.58 1.71
MED29 Mediator of RNA polymerase II transcription subunit 29 0 0 5 104590000 4 258630000 0.40 2.47
MED30S;MED30 Mediator of RNA polymerase II transcription subunit 30 0 0 4 44101000 5 128540000 0.34 2.91
MED31 Mediator of RNA polymerase II transcription subunit 31 0 0 2 42416000 1 36983000 1.15 0.87
CDK8 Cyclin-dependent kinase 8 0 0 1 4874300 0 0 X 0
CDK19 Cyclin-dependent kinase 19 0 0 2 19045000 0 0 X 0
MED12 Mediator of RNA polymerase II transcription subunit 12 0 0 27 351070000 0 0 X 0
MED13 Mediator of RNA polymerase II transcription subunit 13 0 0 19 176110000 6 78870000 2.23 0.45
MED13L Mediator of RNA polymerase II transcription subunit 13-like 0 0 4 21724000 3 24112000 0.90 1.11
Cyclin-C CCNC 0 0 2 50450000 0 0 X 0
20. Conclusions
1. SJSAs have lower amounts of CDK8, MED12 and MED26 than HeLa
2. CDK19 and Cyc C are likely limit of detection issues
4. CDK19 IPs will help with limit of detection issues
a. So will bead-clearing IPs in the future
a. Why?
3. HeLa and SJSA IPs were well-matched for total protein and Mediator
21. Future Directions
• Validate Med26 is actually lower in SJSA by Western
Blot
• Search specific proteins between HeLa and SJSA
• Validate any specific proteins with Western Blot
• Replicates and growth of SJSA cells and experiments
• Eventually complete CDK19-IP experiments with
comparisons