Protein Extraction and Western Blotting Protocol

Protein was extracted from cells using M-PER mammalian protein extraction reagent (78503; Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (P8340; Sigma-Aldrich). Protein samples were mixed with sample buffer (30566-22; Nacalai Tesque, Kyoto, Japan) and heated at 96 °C for 5 min. Western blotting was subsequently performed as previously reported [62 (link)]. When needed, membranes were cut based on the molecular weight to separately hybridize with different antibodies. The primary antibodies were summarized in Supplementary Table S3. The secondary antibody was goat anti-rabbit IgG horseradish peroxidase (1:10,000, 7074; Cell Signaling Technology). The resulting bands were detected using SuperSignal West Pico Chemiluminescent Substrate (34580; Thermo Fisher Scientific) and the ChemiDoc XRS Plus System (Bio-Rad Laboratories). The intensity of the bands was quantified using ImageJ software.