Cortactin

Cells were lysed with RIPA lysis buffer (Beyotime) and added the protease inhibitor. Next, the concentration of proteins was measured by a BCA Protein Quantification Kit (Yeasen). The standard western blotting protocol was followed. The primary antibodies included antibodies specific for EMT markers, Desmoplakin (Proteintech, # 25318-1-AP), ZEB2 (Proteintech, # 14026-1-AP), N-cadherin (Proteintech, # 22018-1-AP), E-cadherin (Proteintech, # 20874-1-AP), Vimentin (Proteintech, # 10366-1-AP), Slug (Signalway Antibody, #24463), Snail (Cell Signaling Technology, # 3879 T), GAPDH (Proteintech, #10494-1-AP), N-WASP (Cell Signaling Technology, # 4848 T), Phospho-N-WASP (Affinity Biosciences, # AF7032), Cortactin (Proteintech, # 11381-1-AP), SF3B3 (Abcam, # ab209402), IKKα (Cell Signaling Technology, #11930), IKKβ (Cell Signaling Technology, #8943), Phospho-IKKα/β (Cell Signaling Technology, #2697), Phospho-p65 (Cell Signaling Technology, #3033), IκBα (Cell Signaling Technology, #4814), Phospho-IκBα (Cell Signaling Technology, #2859), and p65 (Cell Signaling Technology, #8242) were detected. The antibody dilution ratios were determined according to the protocol. The reference gene was GAPDH.

The cultured cell lysates were obtained by RIPA lysis buffer (#P0013B, Beyotime Biotechnology, Shanghai, China) with protease inhibitors (#87786, Thermo Fisher, Waltham, MA, USA). Then, protein was extracted, quantified, separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes. After being blocked with bovine serum albumin, the target protein was probed with antibodies against human E-cadherin (1:1000; #3195, Cell Signaling Technology), ERRα (1:500; #ab137489, Abcam, MA, USA), HMGCS1(1:1000; #17643-1-AP, Proteintech, Wuhan, China), Cortactin (1:4000; #11381-1-AP, Proteintech, Wuhan, China), GAPDH (1:3000; #60004-1-Ig, Proteintech, Wuhan, China), MMP2 (1:2000; #10372-2-AP, Proteintech, Wuhan, China), Vimentin (1:1000; #5741, Cell Signaling Technology), next incubated with secondary antibodies (1:6000; 66031-1-Ig, Proteintech, Wuhan, China), and detected with enhanced chemiluminescence reagents (#34577, Thermo Fisher Scientific, Waltham, MA, USA). Finally, protein bands were imaged by FluorChem M automatic chemiluminescence image analysis system (Protein Simple, San Jose, CA, USA).