Phospho p70s6k

Cells were lysed in PLB buffer (100 mM NaCl, 0.1 M sodium phosphate, pH 7.5, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate). The buffer was supplemented with 1% protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA) and 1 mM PMSF. In experiments assaying levels of phosphorylated proteins, phosphatase inhibitors (1 mM NaF, 1 mM sodium vanadate, 40 mM βGPO₄) were also added. Proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes, which were incubated with Abs to FLAG, LC3B, α-tubulin and actin (Sigma-Aldrich), phospho-p70S6K (Thr389), p70S6K, phospho-4E-BP1 (Thr37/46), non-phospho-4E-BP1 (Thr46), 4E-BP1, phospho-AMPKα (Thr172), AMPKα, phospho-ULK1 (Ser757), phospho-RXRXXpS/T, phospho-MLC (Ser19), mTOR and raptor (Cell Signaling, Danvers, MA, USA), ULK1 (Santa Cruz, Santa Cruz, CA, USA), HA (Biotest, Boca Rato, FL, USA), DAPK2 (Abcam, Cambridge, MA, USA; ProSci). Myc Ab was a gift from M Oren (Weizmann Institute of Science, Rehovot, Israel). Detection was carried out with either HRP-conjugated goat anti-mouse or anti-rabbit secondary Abs (Jackson ImmunoResearch, Suffolk, UK), followed by enhanced chemiluminescence (SuperSignal; Pierce, Thermo Scientific, Rockford, IL, USA). Protein densitometric analysis was performed using EZQuant-Gel software (EZQuant, Tel-Aviv, Israel) on scanned blots.