Whole-cell Lysis and Western Blotting

Whole-cell lysis from treated cells were extracted with RIPA buffer containing 10 mM Tris-Cl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 140 mM NaCl. Lysis products were ultrasonicated and then centrifuged at 12,000 × g for 15 min at 4 ˚C. Protein concentrations were measured using BCA Assay Reagent (#23228, Thermo Fisher Science, Inc.). The western blotting was performed as described previously [15 (link)]. Protein samples (20 μg) were subjected to SDS-PAGE electrophoresis and IB analysis. Briefly, the proteins were transferred to a PVDF membrane and then incubated with 5% skimmed milk. Primary antibodies were incubated overnight at 4 ˚C, and secondary antibody was incubated for 30 min at room temperature. The immunoblot bands were observed with ECL reagents (#34579, Thermo Fisher Scientific, Inc.).