Western Blot Analysis of Immune Signaling

Western blotting analysis was performed as previously described (Wang et al. 2017 (link)). Briefly, total proteins from cultured cells were obtained, separated by SDS–PAGE electrophoresis and transferred onto nitrocellulose membranes. Primary antibodies against IL-1β (1:1000, CST), NLRP3 (1:500, Proteintech), NF-κB (1:1000, CST), p-NF-κB (1:1000, CST), Atp5a1 (1:2000, Proteintech), Ndufv2 (1:5000, Proteintech), β-actin (1:10,000, Proteintech) and appropriate secondary antibodies were used. The optical density of bands was analyzed by ImageJ software. The expression levels of the examined proteins were normalized to those of β-actin.