Horseradish peroxidase hrp conjugated goat anti rabbit igg h l secondary antibody

Total protein was extracted using RIPA lysis buffer with protease inhibitor cocktail (Pierce, USA), and a BCA kit (Thermo Scientific, USA) was employed to quantify protein concentration. 40 μg of protein per specimen was added to each well of 4-12% NuPAGE Bis-Tris precast gels (Invitrogen, CA) for electrophoretic separation of proteins. Proteins were then transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk for 1 h at room temperature, then incubated with different primary antibodies: rabbit anti-SUMO-1 (Abcam, USA), rabbit anti-TP53INP1 (Abcam, USA), mouse anti-p53 (Abcam, USA), rabbit anti-CBX4 (Santa Cruz Biotechnology, USA), rabbit anti-PIAS3 (Abcam, USA), rabbit anti-SENP1 (Abcam, USA), rabbit anti-GAPDH (Abcam, USA), SUMO-1 polyclonal antibody (Abcam, USA), or TP53INP1 polyclonal antibody (Abcam, USA) at 4°C overnight. Horseradish peroxidase- (HRP-) conjugated goat anti-rabbit IgG (H+L) secondary antibody (Promega, USA) and HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (Promega, USA) were then added, and samples were incubated at room temperature for 2 h. The protein bands were visualized using an ECL Western Blotting Substrate kit (Pierce, USA) after which analysis of protein bands was conducted using the ImageJ software. Three independent experiments were performed.