Goat anti rabbit igg secondary antibody conjugated to horseradish peroxidase

Total protein was extracted from cells. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime, Jiangsu, China) and then transferred onto PVDF membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed at room temperature for 2 h. Anti-CLDN8, anti-IL22, anti-p-ERK, anti-ERK, anti-cleaved caspase-3, anti-bax, anti-bcl-2, anti-XIAP, anti-VEGF, anti-MMP-2, anti-E-cadherin, anti-N-cadherin and anti-GAPDH (1:800, abcam) were added overnight at 4 °C. The membranes were subsequently incubated with goat anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (1:5000, abcam) at room temperature for 2 h. Finally, proteins were visualized using a WestrenBright ECL Kit (Advansta, USA).

The Total Protein Extraction kit was applied to the extraction of total protein. SDS-PAGE electrophoresis was used to separate identical quantities of proteins, which were then transferred onto nitrocellulose filter membranes. Primary antibodies specific for STAT1, N-cadherin, Vimentin, E-cadherin, VDAC1 or GAPDH were used to incubate the blots that were blocked with bovine serum albumin overnight at 4°C. GAPDH served as an endogenous control. At room temperature, we used goat anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (1:5000, abcam) to subsequently incubate the membranes for 2 h. All antibodies were purchased from Aviva Technology(Haidian, Beijing, China). An Imaging System was used to scan the bands.