A19714

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).

Immunohistochemistry staining was performed on paraffin sections with rabbit monoclonal antibodies directed against PSMD4 (1:100; Enzo Life Sciences Cat# PAB0242, RRID:AB_1532684), NF-κB p65 (1:100; Cell Signaling Technology Cat# 3034, RRID:AB_330561), and IκBα (1:100, ABclonal Cat# A19714, RRID:AB_2862749). The biotinylated goat anti-rabbit secondary antibody (1:5,000, Signalway Cat# L3012, RRID:AB_895483) was incubated at 4 °C. Finally, the sections were incubated with DAB substrate for 5 min. To validate the PSMD4, NF-κB p65, and IκBα antibodies used in immunohistochemistry experiments, liver cancer and para-cancerous tissue samples were used as positive and negative controls (Figure S1).