Cells were lysed with RIPA buffer (Solarbio, China) containing protease inhibitor cocktail (Sigma-Aldrich). Cell lysates were sonicated on ice, resolved on SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 5% non-fat milk, the membranes were probed with primary antibodies for the detection of WASH (ab157592, Abcam) and β-actin (3700S, Cell Signaling Technology) followed by horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology). Protein expression was detected using enhanced chemiluminescent HRP substrate (Thermo Fisher Scientific) and photographed with a Chemiluminescence Imaging System (Bio-Rad Laboratories).
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