Sodium vanadate

GMDCs were washed in PBS, snap-frozen in liquid nitrogen, and lysed in ice-cold buffer containing: 50 mM Hepes (pH 7.6) (Sigma-Aldrich), 50 mM NaF (Sigma-Aldrich), 50 mM KCl (MilliporeSigma), 5 mM NaPPi (Sigma-Aldrich), 1 mM EDTA (Thermo Fisher Scientific), 1 mM EGTA (Sigma-Aldrich), 1 mM DTT (Promega), 5 mM β-glycerophosphate (Sigma Aldrich), 1 mM sodium vanadate (Sigma-Aldrich), 1% NP40 (Sigma-Aldrich), and protease inhibitors cocktail (Complete, Roche). Proteins were separated by SDS-PAGE and transferred to a PVDF membrane. Membranes were blocked for 1 hour at room temperature in TTBS buffer (20 mM Tris–HCl [pH 7.6; Thermo Fisher Scientific], 137 mM NaCl [MilliporeSigma], and 0.25% [v/v] Tween 20 [MilliporeSigma]) containing 5% (w/v) fat-free milk. Membranes were incubated with primary antibodies overnight at 4°C, washed in TTBS buffer, incubated with horseradish peroxidase–conjugated secondary antibodies for 2 hours at room temperature, washed again, and developed using enhanced chemiluminescence. Primary antibodies included AMPK (Cell Signaling Technology, 2532) and HSP90 (Santa Cruz Biotechnology Inc., sc7947).

An affinity-purified monoclonal anti-FLAG antibody, leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF), and sodium vanadate were obtained from Sigma Chemical Co. (St. Louis, MO) and anti-pAKT, AKT, phosphorylated ERK1/2, ERK1/2, and RasGRP3 antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti-Ras and anti-Rap1 antibodies were purchased from BD Biosciences (San Jose, CA). The AKT kinase-dead mutant vector was obtained from Addgene. The H-Ras dominant negative mutant vector was a gift from Professor Raymond Mattingly from Wayne State University (Detroit, MI). The pFLAG-RasGRP3 with an N-terminal FLAG tag was described previously [48 (link)].

Lysates were prepared by washing embryoid bodies twice in PBS. Ice-cold RIPA buffer was added which contains 150 mM NaCl (Sigma), 50 mM Tris HCl pH 8 (Sigma), 1% NP-40 (VWR), 0.5% Na Deoxycolate (Fisons), 0.1% SDS (Sigma), 25 units/ml Benzonase nuclease (Sigma: E1014), supplemented with either phosphatase inhibitor cocktails 2 and 3 (Sigma: P5726 & P0044) and cOmplete Mini, EDTA-free protease inhibitor cocktail (Roche: 04693159001) or the following protease/phosphatase inhibitors: 1 mM sodium vanadate (Sigma: S-6508), 1 mM sodium molybdate (BDH AnalR: 10254), 10 mM sodium fluoride (Sigma: S6521), 10 µg/ml Phenylmethylsulphonyl fluoride (PMSF) (Sigma: P7626), 0.7 µg/ml Pepstatin A (Sigma: P5318), 10 µg/ml Aprotinin (Roche: 236624), 10 µg/ml Leupeptin (Sigma: L8511), 10 µg/ml Soyabean trypsin inhibitor (Roche: 109886) in H₂O. Samples were kept at −20°C until required. Protein concentrations were determined using the BCA assay (Thermo Scientific) according to the manufacturer’s directions.