Anti p ikkβ

Western blotting was conducted as in prior reports [15 (link)], using the following primary antibodies: anti-NF-κB p65 (Cell Signaling Technology, Danvers, MA; 8242, 1:1000), anti-p-IKKβ (Abcam, ab59195, 1:1000), anti-p-IκBα (Cell Signaling Technology, 14D4, 1:1000), anti-LTβR (Abcam, Burlingame, CA; ab65089, 1:1000), anti-GAPDH (Proteintech, Wuhan, China; Cat No. 60004-1-Ig, 1:1000).

Cell Counting Kit-8 (CCK-8), Bicinchoninic acid (BCA) Protein Assay Kits, Annexin V-FITC Apoptosis Detection Kits, and Cell Cycle Analysis Kits were all purchased from the Beyotime Institute of Biotechnology (Jiangsu, China). Protein Extraction Kits were obtained from KEYGEN Biotech. Co., Ltd. (Nanjing, China). Diaminobenzidine (DAB) substrate kits were purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) kits were provided by Roche Diagnostics (Germany). 4′,6′-Diamidino-2-phenylindole (DAPI), tris (hydroxymethyl) aminomethane (Tris), and sodium dodecyl sulphate (SDS) were purchased from Sigma (St. Louis, MO, U.S.A.). Anti-LOX1, anti-caspase-9, anti-p65, anti-p-IKKβ, anti-IKKβ, anti-p-IkBα, anti-IkBα, anti-GAPDH, anti-β-tubulin, and anti-Lamin B primary antibodies, and horseradish peroxidase-conjugated antibody were obtained from Abcam (Cambridge, U.K.). Plasmids harboring the wild-type caspase-9 response element (WT-luciferase-caspase-9) and the corresponding mutant (MUT-luciferase-caspase-9) were purchased from Vipotion Biotechnology (Guangzhou, China). Pierce Agarose Chip Kits were obtained from Thermo Fisher Scientific (Waltham, MA, U.S.A.).

Subcellular fractionation was performed with a Nuclear and Cytoplasmic Protein Extraction Kit (Sangon, Shanghai, China), in accordance with the manufacturer’s instructions. Total protein separation and western blotting were performed as described previously [30 (link)]. The following antibodies were used: anti-p-IκBα (Cell Signaling Technology, USA), anti-p-IKKβ (Abcam, Cambridge, MA, USA), anti-β-actin, anti-Lamin B1, anti-TRAF1 and anti-NF-κB p65 (Proteintech, Wuhan, China). All experiments were performed in triplicate.