Western Blot Protein Analysis Protocol

Cells or subcellular components were lysed three times with 1× cell lysis buffer (Cell Signaling Technology, 9803) containing protease inhibitor on ice for 10 min. Protein was quantified using a bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, 23225), and 20 to 40 µg of each sample was resolved on 4 to 12% Criterion XT Bis-Tris gels (Bio-Rad, 3450124) in XT MES running buffer (Bio-Rad, 1610789) and transferred to polyvinylidene difluoride membranes (Bio-Rad, 1620233) using the Trans-Blot Turbo Transfer Pack and System. Membranes were blocked with tris-buffered saline with Tween 20 (TBST) containing 5% skim milk for 1 h and incubated overnight at 4 °C with various primary antibodies. Following three washes in TBST, membranes were incubated with goat anti-rabbit or anti-mouse immunoglobulin G (IgG) horseradish peroxidase secondary antibody (Cell Signaling Technology, 7074 or 7076; 1:1000) at room temperature for 1 h and washed. Chemiluminescence substrate was applied using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, 34080) or the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34095), and blots were analyzed using the ChemiDoc Touch Imaging System (Bio-Rad) and Image Lab Software (Bio-Rad, version 6.1)^{77 (link)}. The information on antibodies is shown in Supplementary Table 1.