Horseradish peroxidase labeled secondary antibody igg

Cells were lysed using RIPA buffer and total protein was extracted from the lysate. Total protein concentration was determined using a bicinchoninic acid assay kit (KeyGen, Nanjing, China). Protein samples were separated by electrophoresis on a 13% sodium dodecyl sulfate–polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). After blocking with 5% skim milk at 25 °C for 1 h, the membrane was incubated with the following primary antibodies: anti-Bcl-2 (1:1000; Abcam, USA), anti-Bax (1:1000; Abcam), anti-P-Iκnt (1:1000; Beyotime, China), anti-Iκnt (1:1000; Beyotime, China), anti-P-p65 (1:1000; Abcam), anti-p65 (1:1000; Abcam), and anti-β-actin (1:500; Beyotime, Hangzhou, China) overnight at 4 °C. After washing with Tris-buffered saline containing 0.1% Tween 20 (TBST), the membrane was incubated with horseradish peroxidase-labeled secondary antibody IgG (1:1000; Abcam, USA) for 1 h at 37 °C. Blots were visualized with an enhanced chemiluminescence kit (Amersham, Little Chalfont, UK) and quantified using the Image Pro-Plus 6.0 analysis system (Media Cybernetics, Rockville, MD, USA). Band density values were normalized to β-actin.