P actin

Cell extracts for immunoblot analysis were prepared as previously described (Lin et al., 2011 (link)). Following electrophoresis, proteins were transferred onto nitro-cellulose membranes and probed with either anti-PPP3CA (Cell Signaling Technology, Danvers, Massachusetts) or p-actin (Santa Cruz Biotechnology, Dallas, Texas) antibodies. Bound antibodies were detected using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific).

The cells were washed twice with cold PBS and then harvested for Western blotting. Cells were lysed in cold radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors for 30 min. The lysates were then centrifuged and the supernatants collected. Approximately 40 µg of total protein was denatured and separated by 10% SDS-PAGE, and then transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 hours at room temperature. The membranes were then incubated with the following primary antibodies overnight at 4°C: PCNA (Abcam, USA), pan Cytokeratin/AE1/AE3, Vimentin (Santa Cruz Biotechnology, USA), E-cadherin, p-MEK1/2(Ser217/221), MEK1/2, p-ERK1/2(Thr202/204), p-SAPK/JNK(Thr183/Tyr185), SAPK/JNK, p-p38 MAPK(Thr180/Tyr182), p38 MAPK, ERK1/2 (Cell Signaling Technology, USA), MMP2 and MMP9 (Boster Biological Technology, Wuhan, China). P-actin (Santa Cruz Biotechnology) was used as a loading control. The membranes were washed five times with TBST and then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. The signals were visualized using an Enhanced Chemiluminescence Detection Kit (Pierce Biotechnology, USA).