Hrp conjugated goat anti mouse or anti rabbit igg antibody

Proteins from cell lysates were denatured in SDS and separated with 10% SDS–PAGE before being transferred onto PVDF membranes (Millipore, USA). Nonspecific binding was blocked using 5% nonfat milk in TBST (10 mM Tris–HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.5) for 1 h at room temperature. The membranes were incubated with specific antibodies against β-actin (diluted at 1:1,000, Santa Cruz, CA, USA), CD147 (diluted at 1:1,000, Abcam, Cambridge, UK), STAT1, STAT3, phospho-STAT1, and phospho-STAT3 (diluted at 1:1,000, Cell Signaling Technology, MA, USA) overnight. The membranes were washed with TBST and then incubated with a secondary HRP-conjugated goat anti-mouse or anti-rabbit IgG antibody (diluted at 1:5,000, Santa Cruz, CA, USA).

Protein extracts fractionated by 10% SDS-PAGE and electroblotted onto Immobilion-P polyvinyldifluoride membranes (Bio-rad) were probed with antibodies against HP1 (1:500, 9A9 monoclonal mouse), α-Tubulin (mouse 1:2000, Sigma). Proteins of interest were detected with HRP-conjugated goat anti-mouse or anti-rabbit IgG antibody (1:10000, Santa Cruz) and visualized with the ECL Western blotting substrate (GE Healthcare), according to the provided protocol. The chemiluminescence detection was performed on the ChemiDoc XRS+ System (Bio-rad) and analyzed using the included ImageLab software.