Mini protean tris glycine gels

Cells were lysed in RIPA buffer (150 mM NaCl, 1% IGEPAL CA-630, 0.5% of sodium deoxycholate, 0.1% sodium dodecylsulfate [SDS], and 50 mM Tris) supplemented with protease inhibitor cocktail (Biovision, Milpitas, CA). Total protein concentration of the lysates was measured using bicinchoninic acid (BCA) assay (Thermo Fisher) with BSA as a standard. Proteins were resolved via SDS–PAGE in 10% Mini-PROTEAN Tris-glycine gels (Bio-Rad, Hercules, CA) and wet transferred to 0.45 μm nitrocellulose membrane (GE Healthcare). Membranes were blocked with either 5% milk dissolved in Tris buffered saline (TBS) or Intercept TBS blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 h at room temperature, then incubated overnight at 4°C with primary antibodies diluted in the blocking solution containing 0.1% Tween-20. Membranes were washed in TBS, 0.1% Tween-20 (TBST) and incubated with HRP- or IRDye-conjugated secondary antibody diluted at 1:5000 in blocking solution containing 0.1% Tween-20. After washing, protein signals were detected via enhanced chemiluminescence (Western Lightning Plus ECL; Perkin Elmer, Waltham, MA) followed by exposure to film (CL-XPosure; Thermo Fisher). For quantitative blots, protein signals were detected on LI-COR Odyssey CLx and quantified using Empiria Studio software.