Immuno blot membrane

Mouse tissues or cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and protein concentration was measured. Total proteins were resolved by SDS-PAGE, transferred to Immuno-Blot membrane [poly(vinylidene difluoride)]; Bio-Rad, Hercules, CA), and immunoblotted with indicated antibodies. Immunocomplexes were visualized with an enhanced chemiluminescence detection kit (Amersham, Piscataway, NJ), using horseradish peroxidase–conjugated secondary antibodies (Cell Signaling).

Total protein was extracted using RIPA lysis buffer (Cat #9806, Cell Signaling, Danvers, MA, USA) with the addition of 1:100 protease PMSF inhibitors (Cat # 5872S, Cell Signaling, Danvers, MA, USA). When running the Western blot, equal quantities of proteins were loaded into each well and separated by size on a polyacrylamide SDS gel. Then, the proteins were transferred to a PVDF membrane (BIO-RAD Immuno-Blot^® Membrane). Once the transfer was complete, nonspecific binding was blocked by incubating the membrane on a shaker with a blocking buffer comprised of 10% skimmed milk in TBS for 1 h at room temperature. The membranes were then incubated with a primary antibody (Supplementary Table S1) at 4 °C overnight. This was followed by incubation with either anti-rabbit IgG or anti-mouse IgG secondary antibody conjugated to HRP horseradish peroxidase (Cell Signaling, Danvers, MA, USA) in antibody dilution buffer for 1 h at 37 degrees Celsius. Washing steps were performed after primary and secondary antibody incubations, which involved 3 washes with TBS buffer + 1% Tween for 5 min each. The ECL substrate Chemiluminescence signal was detected using the ChemiDoc imaging system (Bio-Rad Laboratories, Hercules, CA, USA).