Bolt mes running buffer

Cells were harvested and washed three times in 1× PBS and lysed in radioimmunoprecipitation assay buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 1% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS, and 500 mM DTT] supplemented with aprotinin (1 mg/ml), leupeptin (1 mg/ml; Sigma-Aldrich), and pepstatin (1 mg/ml; BMB). Whole-cell lysate (50 mg) was loaded in Bolt 4 to 12% Bis-Tris Plus gel (Invitrogen) or Novex WedgeWell 10% tris-glycine gel (Invitrogen) and separated through gel electrophoresis [SDS–polyacrylamide gel electrophoresis (SDS-PAGE)] in Bolt MES running buffer (Invitrogen) or tris-glycine-SDS buffer (Bio-Rad), respectively. Separated proteins were transferred to ImmunBlot polyvinylidene difluoride membranes (Bio-Rad) for antibody probing. Membranes were incubated with 10% bovine serum albumin (BSA) in tris-buffered saline containing 0.1% tween (TBST) for 30 min at room temperature, then incubated for 2 hours at room temperature (RT) or overnight at 4°C with the suitable antibodies diluted in 5% BSA in 1× TBST, washed with TBST, and incubated with a dilution of 1:10,000 of horseradish peroxidase–linked anti-mouse or anti-rabbit secondary antibody (Cell Signaling Technology) for 1 hour at RT. Antibodies were then visualized using Clarity Western ECL substrate (Bio-Rad) and imaged using Fujifilm LAS-3000 Imager (Fujifilm).