Vimentin

At the endpoint of treatment, cultured cells were lysed with RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor. The supernatant was extracted for the measurement of total protein with a protein assay kit (BCA). Samples were dissociated with SDS-PAGE and then transferred to PVDF membranes and incubated with primary antibody for GAPDH [Kangchen Biotech (Shanghai, China), Cat# KC-5G4, RRID:AB_2493106], α-SMA [Cell Signaling Technology (Beverly, MA, USA), Cat# 19245, RRID:AB_2734735], SM22α (Cell Signaling Technology, Cat# 52011), Vimentin [Solarbio (Beijing, China), Cat# GB11192, RRID:AB_2814685], P65 Cell Signaling Technology Cat# 8242S), acetyal-P65 (Cell Signaling Technology Cat# 12629S), and VCAM-1 (Cell Signaling Technology Cat# 39036, RRID:AB_2799146) at 4 °C overnight. The membranes were washed 3 times and incubated for 1 h at room temperature with horseradish-peroxidase-(HRP)-conjugated secondary antibodies. Images were quantified using the digital gel image analysis system TANON 5500, and ImageJ Version 1.46r analysis software was used for protein expression analysis.