Western Blot Analysis Protocol

Western blot analysis was performed according to the protocol of our previous study (Son et al., 2020 (link)). The samples for SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) were prepared by extracting proteins from 3T3‐L1 cells with radio‐immunoprecipitation assay buffer (Boston Bio Products) containing protease inhibitor (Sigma‐Aldrich) and phosphatase inhibitor (Sigma‐Aldrich) and quantifying the protein amounts by BCA assay (Thermo Fisher Scientific). After sample preparation, the protein (25 μg/well) was separated by SDS‐PAGE, and then proteins separated on the gel were transferred to the polyvinylidene fluoride (PVDF) membrane. After the transfer, the PVDF membrane was blocked with 5% nonfat milk in Tris‐buffered saline containing .05% Tween 20 (TBS‐T) at room temperature for 1 hr, and then, the PVDF membranes were treated with the primary antibodies in 5% bovine serum albumin in TBS‐T at 4ºC for overnight. After the primary antibody treatment was completed, the PVDF membranes were treated with the secondary antibodies in 5% nonfat milk in TBS‐T at room temperature for 1 hr. Chemiluminescence was detected with ECL Western blotting substrate (Amersham Biosciences) and visualized using LI‐COR C‐DiGit Blot Scanner (Li‐COR Biosciences). The density of Western blot bands was calculated using the software UN‐SCAN‐IT gel version 5.1 (Silk Scientific Inc.).