1. Summer Scholarship 2014 Outcomes
Rose Upton. 3163409.
Overview:
The aim of this project was to investigate FZR1, an activator of the anaphase promoting
complex/cyclosome and regulator of mitotic cell division. By using Immunohistochemistry (IHC), Western
Blotting and Immunoprecipitation (IP), possible protein targets of FZR1 were analysed for interactions with
FZR1. Specifically, changes in protein localisation from wild type and FZR1 knockouts in postnatal mice
(Days 6, 11 and 18) were examined using the listed techniques.
Protein Phosphatase 2A catalyticsubunit α (PP2A-α):
Using Immunohistochemistry, the localisation of PP2A-α was investigated in FZR1 wild type and knockout
histological sections of postnatal day 6 and day 11 testis of mouse. For initial optimisation, two antibodies
for PP2A-α from different suppliers (NB100-9227 and BD 61055) were tested with sodium citrate antigen
retrieval and an alexa fleur 594 secondary. The most significant staining was seen within the NB100-9227
antibody with no staining appearing in either of the day 6 sections or the wild type day 11, but large
amounts of localisation within the day 11 knockout.
To determine whether these results were correct, IHC was repeated using the NB100-9227 PP2A-α
antibody on a second animal set of histological sections. Similar results were yielded, however a large
amount of protein was seen in both the day 11 wild type and the knockout. To see which result was
correct, a third set of animals was used for another repeat. These results differed from the previous results
significantly with localisation of the protein identified within the knockout of day 6 only.
Three repeats of IHC on each animal set were completed, including three dual stains using tyrosine tubulin
(T9028). Each repeat within animal sets yielded consistent results. As a western blot was completed to
confirm the presence of the protein within the tissue it was concluded that PP2A-α was too variable and
investigations into this line of enquiry were discontinued.
Cell Division Cycle 14B (CDC14B):
Using Immunohistochemistry, the localisation of CDC14B was investigated in FZR1 wild type and knockout
histological sections of postnatal day 6 and day 11 testis of mouse. The antibody used was H00008555-A01
and antigen retrieval was achieved via sodium citrate. The first set of IHC was carried out on histological
sections labelled animal set 3 for the purpose of investigations using PP2A-α.
Localisation was identified in all sections with perhaps a slight increase in staining for the knockout day 6
when compared to wild type. Within the day 11 sections overall intensity of staining was higher within the
wild type section, however within the knockout section some tubules had a much more pronounced
localisation than others. This result was seen again using animal set 2.
From here it was decided to complete IHC on postnatal day 18 histological sections of FZR1 wild type and
knockout testis of mice. Once again the difference between wild type and knockout sections was
imminent. A western blot was completed using day 6 and day 11 wild type and knockout protein proving
the presence of the protein.
The next step of the investigation into CDC14B involved an immunoprecipitation, in which an interaction
between CDC14B and FZR1 could be determined. The results did not reveal an interaction, however further
attempts may be made to optimise the process before concluding that no interaction is evident. Also to be
completed in future study is a western blot using protein from day 18 testis.

2. NEDD4-like E3 ubiquitin-protein ligase 2 (NEDL2):
Using Immunohistochemistry, the localisation of NEDL2 was investigated in FZR1 wild type and knockout
histological sections of postnatal day 6 and day 11 testis of mouse. The antibody used was ab154888 and
antigen retrieval was achieved via sodium citrate. This yielded poor staining that could only be seen at high
sensitivity.
To optimise IHC methods of this antibody a combination of staining techniques, and antigen retrievals as
well as differently fixed sections were tested. Bouins fixed day 11 wild type and knockout sections were
used using alexa fleur 594 as per the usual protocol with one set undergoing sodium citrate retrieval and
the other undergoing TE buffer antigen retrieval. Paraformaldehyde fixed adult wild type sections were
also tested with sodium citrate retrieval. One set of the adult sections was stained using an alexa fleur 594
secondary while the other underwent a VectaStain DAB protocol.
The best results were seen using TE buffer antigen retrieval in the Bouins fixed day 11 wild type sections
with little to no localisation of protein identified within the other sections. Further research here requires a
repeat of TE buffer antigen retrieval in day 6 and day 11 (and possibly day 18) sections using NEDL2 to
determine if there is a difference from wild type to knockout. A western blot should also be completed
with the possibility of progression to an IP, pending results.