Phosphorylated p70s6k

Antibodies against Akt, phosphorylated Akt, PI3K, phosphorylated PDK1, phosphorylated TSC2, phosphorylated GSK3β, phosphorylated p70S6K, phosphorylated 4EBP1, mTOR, PTEN, FOXO1, PCNA, and cleaved poly ADP ribose polymerase (C-PARP) were from Cell Signaling Technology (Danvers, MA). Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), survivin, CDK4, and pRb were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phosphorylated mTOR and phosphorylated PTEN were from Abcam (Cambridge, UK). Antibody against Cyclin D1 was from Biosource (Camarillo, CA).

Cell lysate was subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The protein was transferred to a nitrocellulose membrane and hybridized with antibody against AMPKα (catalog number: #2532), phosphorylated AMPKα at Thr 172 (#2535), 4E‐BP1 (#9452), phosphorylated 4E‐BP1 at Thr 37/46 (#9459), p70S6K (#9202), phosphorylated p70S6K at Thr389 (#9205), phosphorylated p53 at Ser 15 (#9284), AKT (#9272), phosphorylated AKT at Ser 473 (#9271), ACC at Ser 79 (#3661), actin (#4970) (Cell Signaling, Danvers, MA, USA), phosphorylated H2AX at Ser 139 (#613401) (BioLegend, San Diego, CA, USA), p53 (Ab‐6, clone DO‐1) and tubulin‐α (clone DM1A, Thermo Fisher Scientific, Fremont, CA, USA) followed by an appropriate second antibody. The membranes were developed with the ECL system (GE Healthcare, Buckinghamshire, UK). actin and tubulin‐α were used as a loading control. DMSO, a solvent of nutlin‐3a, was used as a control.

Cells were grown to 80% confluence and harvested in lysis buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mN Na₃VO₄, 100 mM NaF, 10 mM NaPPi, 10% glycerol, 1% Triton X-100 (Tx-100)). Lysates were spun at 10,000 RPM for 10 min at 4 °C. Tx-100 soluble cell lysates were subject to BCA protein assay (Pierce, ThermoFisher Scientific, Waltham, MA, USA) and 30 μg protein/sample was separated by SDS-PAGE, transferred to a nitrocellulose membrane, incubated for 1 h in PBS + 5% bovine serum albumin, and analyzed by Western blot with antibodies against the following targets: Phosphorylated p70S6K (1:1000 in TTBS; Cell Signaling Technologies, #9206; Danvers, MA, USA); total p70S6K (1:500 in TTBS; Cell Signaling Technologies, #9202; Danvers, MA, USA); beta-actin (1:1000 in TTBS; Sigma-Aldrich, #A5316; St. Louis, MO, USA). Membrane was incubated with primary antibody overnight. HRP-conjugated secondary antibodies (1:10,000; ThermoFisher Scientific, Waltham, MA, USA) and ECL reagent (ThermoFisher Scientific, Waltham, MA, USA) were used for visualization with a CCD camera imaging system (UVP). Spot densitometry was used to quantify relative signal intensity.

Cells were appropriately treated, and total cell lysates and nuclear extracts were prepared as previously described [35 (link)]. Total protein lysates from cells were prepared by sonicating cells in lysis buffer (iNtRON, Korea). Equal amounts of lysates were boiled at 100°C and resolved by 10-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA) and then blocked with 5% milk in Tris-buffered saline and 0.1% Tween 20. The membranes were incubated overnight with primary Abs against LC3, AMPKα, phosphorylated AMPKα, p70S6K, phosphorylated p70S6K, and β-actin (all from Cell Signaling Technology, Danvers, MA), and proteins were detected with goat-anti-rabbit Abs conjugated with horseradish peroxidase (HRP) (Cell Signaling Technology, Danvers, MA). Proteins were detected using the enhanced chemiluminescence (ECL) method with femtoLUCENT ™ PLUS-HRP (G-biosciences, St. Louis, MO).