Western Blot Analysis Protocol

Western blot analysis was conducted as described before (Dong et al., 2016 (link), 2017 (link)). The cells, seeded in 6-well plates and have been treated or not, were lysed with RIPA lysis solution (Boster) containing broad spectrum phosphatase inhibitors and PMSF (Boster). Total concentration of proteins was measured by BCA assays (Boster, Wuhan, China). The electrophoresis of proteins was conducted in 10% SDS-polyacrylamide gel and then the proteins were transferred to the PVDF membranes (Millipore, MA, United States). 5% bovine serum albumin was used for blocking of the membranes, then the membranes were incubated with respective antibodies overnight. After washed, the membranes were incubated at 25°C for 1 h with horseradish peroxidase-conjugated secondary antibodies (Boster). Subsequently, enhanced chemiluminescence (Boster) was used to visualize the proteins, and images were acquired through ChemiDoc^TM XRS+ System with Image Lab^TM Software (Bio-Rad Laboratories, CA, United States).