A02177 4

Samples were lysed in cell lysis buffer for western and IP (Beyotime). Proteins were quantified by BCA protein assay kit (Beyotime). After complete separation by electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Then these membranes were blocked in 5% skimmed milk (Inner Mongolia Yili Industrial Group Co., Ltd., Inner Mongolia, China) for 1 h and incubated with primary antibodies. Then these membranes were incubated with HRP-labeled goat anti-rabbit IgG (H+L) (1: 5,000, A0208, Beyotime) or horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (H+L) (1: 5,000, A0216, Beyotime) at room temperature at 37°C for 45 min. Protein bands were visualized with Gel-Pro-Analyzer software (Beijing Liuyi Biotechnology Co., Ltd., Beijing, China).
The primary antibodies used were shown as following: HDAC9 antibody (1: 500, A02177-4, BOSTER, Wuhan, China), cleaved caspase-3 antibody (1:1,000, AF7022, Affinity, Jiangsu, China), Bax antibody (1:1,000, A19684, ABclonal, Wuhan, China), B-cell lymphoma/leukemia-2 (Bcl-2) antibody (1:1,000, A19693, ABclonal), IκBα antibody (1:1,000, A19714, ABclonal), p-IκBα antibody (1:1,000, AP0707, ABclonal), p-p65 antibody (1:1,000, AP0123, ABclonal), p65 antibody (1:1,000, A2547, ABclonal), β-actin (1:1,000, sc-47778, Santa Cruz, Santa Cruz, CA, USA).

Cells and tissue slides were incubated with p65 antibody (1:100, A2547, ABclonal) and HDAC9 antibody (1:100, A02177-4, BOSTER) at 4°C overnight, respectively. Then, the slides were incubated with goat anti-rabbit IgG (1:200, A27039, Invitrogen). The fluorescence was captured on a microscope system (Olympus, Tokyo, Japan). Three slides were taken from each group, and one field of view was selected for each slide. The number of p65 (+) cells in the nucleus was calculated by counting of each visual field.