Immunoblotting Assay for Apoptosis Markers

The protein levels of cyclin D1, ki67, caspase-8, caspase-3, cleaved-caspase-8, cleaved-caspase-3, RIPK1, IKKβ, p-IκBα, and p-p65 were examined by immunoblotting following the methods described before (36 (link)) with antibodies against cyclin D1 (60186-1-Ig; Proteintech, Wuhan, China), ki67 (27309-1-AP, Proteintech), caspase3 (19677-1-AP, Proteintech), cleaved-caspase 3 (ab2302, Abcam, Cambridge, MA, USA), caspase-8 (ab32397, Abcam), cleaved-caspase-8 (# 9496S; Cell Signaling; Danvers, MA, USA), IKKβ (07-1008; Sigma-Aldrich, St. Louis, MO, USA), p-IκBα (#9246; CST, Danvers, MA, USA), p-p65 (ab194726, Abcam), and β-actin (60008-1-Ig, Proteintech). β-actin was taken as an endogenous control. The incubation of membranes with primary antibody was followed by another incubation with HRP-conjugated secondary antibodies. Protein blots were then visualized using enhanced chemilumescent (ECL) substrates (Millipore, MA, USA).