Phospho rps6 ser235 236

MC3T3-E1 cells were lysed with TNE buffer containing 50 mM Tris (pH 7.4), 140 mM NaCl, 5 mM EDTA and a protease inhibitor tablet (Roche), followed by sonication. Protein concentration of lysates were determined using Bradford reagent (AMERSCO). Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Pall corporation), incubated with blocking buffer (5% non-fat dry milk in Tris-buffered saline and 0.1% Tween 20) for 30 minutes. Immunoblots were performed using primary antibodies against NFIL3, hnRNP A1, 14-3-3ζ, LAMIN B (Santa Cruz), Phospho-RPS6 (Ser 235/236) (Cell Signaling), ACTIN (MPBIO), GAPDH (Millipore) and FLAG (Sigma-Aldrich). Horseradish peroxidase (HRP)-conjugated mouse (Thermo Scientific), rabbit (Promega), rat or goat (Bethyl Laboratories) secondary antibodies were detected with SUPEX ECL reagent (Neuronex) and a LAS-4000 system (FUJI FILM), according to the manufacturer’s instructions. The integrated blot density was quantified through Image J software-based analysis (http://rsb.info.nih.gov/ij/).

S6K1, phospho-S6K1(Thr389), rpS6, phospho-rpS6(Ser235/236), IRE1α, CHOP, GADD34, ATF4, TSC2, SESN2, AMPK, phospho-AMPK(Thr172), ACC, phospho-ACC (Ser79) and β-actin antibodies were purchased from Cell Signaling Technology (Danvers, USA). LC3 antibodies were bought from Novus Ltd. (Cambridge, UK). Cells were washed in ice-cold phosphate buffered saline (PBS) and then lysed in cell lysis buffer (20 mM Tris (pH 7.5), 125 mM NaCl, 50 mM NaF, 5% (v/v) glycerol, 0.1% (v/v) Triton X-100, supplemented with 1 mM dithiothreitol (DTT), 1 µg/mL pepstatin, 20 µM leupeptin, 1 mM benzamidine, 2 µM antipain, 0.1 mM PMSF, 1 mM sodium orthovanadate and 1 nM okadaic acid prior to cell lysis). Cell lysates were sonicated using a diagenode bioruptor (Diagenode, Seraing, Belgium) and centrifuged at 13,000 rpm for 8 min. Protein concentrations were determined by a Bradford assay (Thermo Fisher Scientific, Paisley, UK). Samples were diluted in 4 × NuPAGE loading sample buffer (Life Technologies) with 25 mM DTT and boiled at 70 °C for 10 min. Western blot was performed as previously described [34 (link)].

Cells were rinsed once with ice-cold PBS and lysed in ice-cold lysis buffer (buffer A: 50 mM HEPES-KOH (pH 7.4), 2 mM EDTA, 10 mM pyrophosphate, 10 mM β-glycerophosphate, 40 mM NaCl, 1% Trition X-100 and one tablet of EDTA-free protease inhibitors (Roche) per 25 mL). The soluble fraction of the cell lysate was isolated by centrifugation at 12,000 g for 10 min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using NuPAGE Novex 4%–12% Bis-Tris precast gels (Invitrogen). Western blotting was performed per standard protocol using a nitrocellulose (Whatman, Protran) membrane. The following antibodies were used: mTOR (Cell Signaling Technology, #2972), phospho-mTOR (Ser2448) (Cell Signaling Technology, #2971), phospho-S6K1 (Thr389) (Abcam company, ab126818), S6K1 (Cell Signaling Technology, #9202), phospho-RPS6 (Ser235/236) (Cell Signaling Technology, #2211), RPS6 (Santa Cruz Biotechnology, sc-74459), α-tubulin (Sigma Inc., T6199), phospho-Akt (Ser473) (Cell Signaling Technology, #4060), Akt (Cell Signaling Technology, #9272), PTEN (Cell Signaling Technology, #9188). Secondary antibodies were HRP linked, anti-rabbit IgG (from donkey) and anti-mouse IgG (from sheep), (GE Healthcare, NA934V, NA931V, and NA935V, respectively).