Pt389 p70s6k

Antibodies used in this study were against α-tubulin (Santa Cruz), pT389-p70S6K, p70S6K, ATF4 (Cell Signalling), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Abcam). Horseradish peroxidase-conjugated donkey anti-mouse IgG and anti-rabbit IgG were used as Western blot secondary antibodies (Millipore).

Protease inhibitor cocktail (Complete mini EDTA-free protease inhibitors, Roche, 04693159001), protein phosphatase inhibitor cocktail (Phosphostop phosphatase inhibitors, Roche, 04906837001), anti-mTOR (T-2949) for immunoprecipitation and Histopaque-1077 were purchased from Sigma/Millipore. Anti-mTOR (SC-517464) for Western blotting, -pS²⁴⁴⁸mTOR (SC-293133), -pS²⁴⁸¹mTOR (SC-293089), -Rictor (SC-271081), -Raptor (SC-81537), -p70S6Kα (SC-8418, SC-393967), -Akt1 (SC-5298, SC-55523), -pS⁴⁷³Akt (SC-514032), -FLNA (SC-58764, SC-17749, SC-271440), -IRβ (SC-09), and -PTEN (SC-7941, Cell Signaling #9559) were purchased from Santa Cruz Biotechnology. Anti-pS²¹⁵²FLNA antibody (TA313881) was purchased from Origene Technologies. Anti-pT¹¹³⁵Rictor (#3806) and -pT³⁸⁹p70S6K (#9205) were purchased from Cell Signaling Technology. Insulin (human recombinant; #12585014), zinc solution (#12585014), covalently conjugated protein A/G-agarose beads (#20423), PageRuler™ Plus Prestained Protein Ladder, 10–250 kDa (#26619) and SuperSignal™ West Pico PLUS Chemiluminescent Substrate (#34577) were purchased from ThermoFisher Scientific.

The cells were rinsed two times with ice-cold PBS and scraped and lysed with Giordano buffer (50 mM Tris-HCl pH = 7.4, 250 mM NaCl, 0.1% Triton X-100, 5 mM EDTA, supplemented with phosphatase and protease inhibitors). Equal protein amounts were separated on SDS-PAGE and blotted on PVDF membranes (Millipore, Darmstadt, Germany). The membranes were blocked with 10% non-fat dry milk in TBST (10 mM Tris-HCl pH = 8.0, 150 mM NaCl, 0.2% Tween-20) and incubated with the primary antibodies diluted in 5% BSA/TBST + phosphatase inhibitors overnight at 4 degrees. The membranes were washed with TBST and incubated with HRP-conjugated secondary antibodies (Jackson Laboratories, Bar Harbor, ME, USA). The chemiluminescent signal was visualized using a Chemidoc machine (Biorad, Hercules, CA, USA).
Primary antibodies: pS473-AKT (#4060), AKT (#2920), pT389-p70S6K (#9205), p70S6K (#2708), pS2056- DNA-PKcs (#68716), IGF1Rβ (#9750) were purchased from Cell Signaling Technology Beverly, MA, USA; vinculin (V9131) was purchased from Sigma-Aldrich, St Louis, MO, USA; DNA-PKcs (sc-390849) and p53 (sc-126) were purchased from Santa Cruz Biotechnology, Dallas, TX, USA; and USP7 (A300-0330A-M) was purchased from Bethyl laboratories, Montgomery, TX, USA.