Horseradish peroxidase labeled appropriate secondary antibody

Cells were first lysed with adioimmunoprecipitation (RIPA; Sangon Biotech) lysate mixed with phenylmethanesufonyl fluoride (PMSF; Sigma-Aldrich). Then the supernatant was collected by means of centrifugation, and protein concentrations were measured with the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Afterward, 25 μg of protein was subjected to a 12% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membranes were blocked with TBST (0.1% Triton in PBS) containing 5% nonfat milk for 1 h. Then the membrane was incubated with primary antibodies to INPP4A, P27, P21, and GAPDH (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax, Bcl-2, caspase 3, E-cadherin, N-cadherin, Snail, and vimentin (1:100 dilution; Invitrogen) overnight at 4°C. The membrane was then probed with horseradish peroxidase-labeled appropriate secondary antibody (1:2,000 dilution; Santa Cruz Biotechnology) at room temperature for 1 h. After incubation with a chromogenic substrate, the protein bands were detected using the enhanced chemiluminescence (ECL) method. GAPDH served as the internal control to normalize the expression of target proteins.

Cells were lysed with radioimmunoprecipitation assay (RIPA; Sangon Biotech, P.R. China) containing phenylmethanesufonyl fluoride (PMSF; Sigma-Aldrich). After centrifugation at 12,000 bmp for 10 min, the supernatant was collected and the protein concentration was detected by the BCA protein assay kit (Pierce, Rockford, IL, USA). An equal amount of extracts was subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Darmstadt, Germany), and probed with primary antibodies to INPP4A, GSK3β, Bax, Bcl-2, pro-caspase 3, cleaved-caspase 3, c-Myc, cyclin D1, P27, β-catenin, p-β-catenin, and GAPDH (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. Followed by exposure to horseradish peroxidase-labeled appropriate secondary antibody (1:5,000 dilution; Santa Cruz Biotechnology) for 2 h, the blots were incubated with a chemiluminescent substrate kit (Pierce) and then detected using the enhanced chemiluminescence (ECL) method. GAPDH served as the internal control.