Western Blotting Protocol for Protein Analysis

Western blotting was performed as previously described^{18 (link)}. Briefly, cells were lysed with RIPA buffer containing protease and phosphatase inhibitors for 30 min on ice, and the lysates were centrifuged at 14,000 rpm for 30 min at 4 °C. The protein concentrations were measured by using a BCA Protein Assay Kit (CWBIO, CW0014S) according to the instructions provided. A 5× sodium dodecyl sulfate loading buffer was added to the protein lysates, and the mixtures were boiled for 5 min. Then, the proteins were separated via polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, IPVH0010). The membranes were blocked with 5% nonfat milk for 1 h and incubated with primary antibodies at 4 °C overnight. Then, the membranes were washed with Tris-buffered saline-Tween (TBST) solution and incubated with HRP-conjugated secondary antibodies (1:3,000, Boster, BA1050 & BA1054) for 1 h. Finally, the membranes were washed with TBST solution and detected by using Immobilon Western Chemiluminescent HRP Substrate (Millipore WBKLS0500). The details of the primary antibodies are provided in the supplementary materials.