p70 S6 kinase or p70S6K is a serine/threonine kinase that acts downstream of PIP3 and phosphoinositide-dependent kinase-1 in the PI3 kinase pathway. As the name suggests, its target substrate is the S6 ribosomal protein. Phosphorylation of S6 induces protein synthesis at the ribosome. The phosphorylation of P70S6K at threonine 389 has been used as a hallmark of activation by mTOR and correlated with autophagy inhibition in various situations. However, several recent studies suggest that the activity of P70S6K plays a more positive role in the increase of autophagy. To purchase this antibody use the following link: http://www.stjohnslabs.com/p70-s6-kinase-antibody?filter_name=STJ31332

1. HOW DID OUR
ANTIBODY PERFORM?
CUSTOMER REVIEW: p70 S6 Kinase Polyclonal Antibody (STJ31332)

2. WHAT WAS USED?
Primary antibody: p70 S6 Kinase Polyclonal
Antibody (STJ31332)
Provider: St John's Laboratory
Dilution ratio: 1:200
Application: Immunofluorescence
Materials for validation: HEK293 cells

3. WHAT WAS THE PROTOCOL?
1. HEK293 cells were seeded at a density of 1.0 x 105 cells on 35mm glass bottom dishes for 72
hours incubated in MEM (1X) media supplemented with 10% FBS, 1% L-glutamine, 1%
penicillin-streptomycin and 1% sodium pyruvate at 37°C, 5% CO2.
2. Media was removed and cells washed with PBS (1X).
3. 4% paraformaldehyde was added to each dish and incubated at room temperature for 15 mins.
4. Paraformaldehyde was removed and cells were washed three times with PBS (1X).
5. Permeabilisation solution (0.3% Triton X-100/PBS/0.1% BSA) was added to each dish and
incubated for 5 minutes at room temperature.
6. Permeabilisation solution was removed and cells were washed three times with PBS (1X). Cells
were blocked with blocking buffer (1XPBS/5% BSA) for 30 minutes at room temperature.
7. Blocking buffer was removed and cells were incubated with 1:200 dilutions of primary
antibodies for 1 hour at room temperature.
8. Primary antibody was removed and cells were further incubated with 1:500 dilutions of
secondary antibody, in dark.
9. Secondary antibody was removed and cells were washed twice with PBS (1X) and kept in fresh
PBS.
10. Cells were imaged using confocal microscopy.

4. WHAT DID THE CUSTOMER THINK?
Testing results were provided by Abdullah Ahmed from
Science & Technology Facilities Council, Oxford. They
stated that “In agreement with literature on S6K1, this anti-
S6K1 (STJ31332) antibody has been suitable for
immunofluorescence labelling in our laboratory.”

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the opportunity to test the product’s
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