Zb 2308

Cultured cells in an exponentially growing phase and frozen tissue samples were extracted with 1 ml of lysis buffer. The protein concentration of the lysate was determined by the BCA method (KeyGen, China). The extracts containing approximately 30 μg of protein were loaded onto 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrophoresed, and then transferred to polyvinylidence difluoride (PVDF) membranes (Immobilon-P. 0.45 μm, Millipore Corp., Bedford, MA, USA). The membranes were blocked with 5 % skimmed milk in PBST (phosphate-buffered saline with 0.1 % Tween 20) for 1 h and then incubated with primary antibody of rabbit anti-ECM1 antibody (Protein Group Inc, USA), rabbit anti-Vimentin antibody (ab92574, Abcam, USA), rabbit anti-E-cadherin antibody (ab15148, Abcam, USA), and rabbit anti-β-actin (ab133626, Abcam, USA) antibody at 4 °C overnight. After being washed three times with PBST buffer, the membranes were incubated with secondary peroxidase-conjugated antibody (ZB-2308, ZSGB-BIO) for 1 h at room temperature. After being washed three more times, the protein bands were detected by enhanced chemiluminescence (Pierce, Rockford, USA) according to the manufacturer’s instruction. β-actin antibody was served as a control to confirm equal loading. Densitometry index analysis of the bands was made using gel imagery system.

Monoclonal anti-Actin Beta (ACTB) antibody (AC026, ABclonal Technology, Wuhan, China), monoclonal anti-GFP antibody (AE012, ABclonal Technology), monoclonal anti-RFP antibody (AE020, ABclonal Technology), monoclonal anti-His antibody (AE003, ABclonal Technology), polyclonal anti-phospho-AKT antibodies (AP0140, ABclonal Technology), polyclonal anti-phospho-FOXO antibodies (AP0684, ABclonal Technology), mouse control IgG (AC011, ABclonal Technology), rabbit control IgG (AC005, ABclonal Technology), Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit IgG light chain (AS061, ABclonal Technology), polyclonal anti-AKT antibodies (WL0003b, Wanleibio, Shenyang, China), polyclonal anti-FOXO antibodies (WL02891, Wanleibio), Alkaline phosphatase (AP)-conjugated horse anti-mouse IgG (ZB2310, ZSGB-BIO, Beijing, China), and AP-conjugated goat anti-rabbit IgG (ZB2308, ZSGB-BIO).