P pkcα

Cell lysates were prepared using RIPA lysis buffer (Thermo Scientific) containing a protease inhibitor cocktail (Thermo Scientific) and protein concentration was measured using the BCA assay (Thermo Scientific). An amount of 5 μg total protein was resolved by SDS–polyacrylamide gel electrophoresis and transferred to the nitrocellulose membrane (Bio-Rad). Immunoblotting was performed using 1:1,000 dilutions of the following antibodies, p-PKCα (sc-12356, Santa Cruz Biotechnology), PKCα (sc-208, Santa Cruz Biotechnology), PRDX6 (13585-1-AP, ProteinTech), PLCβ4 (sc-404, Santa Cruz Biotechnology), STX12 (Syntaxin 12) (sc-368438, Santa Cruz Biotechnology) and β-actin (3700S, Cell Signaling).

The tissue fragments were lysed in radioimmunoprecipitation assay buffer and then centrifuged for 5 min at 12,000 × g. The lysate was collected, and the protein concentration was measured using a test kit. Equal amounts of protein were denatured and separated on 10% SDS‒PAGE gels and then transferred to polyvinylidene difluoride membranes. When we performed WB, the blots were cut prior to hybridization with antibodies, and we developed strips of different proteins on the same PVDF film separately. The membranes were blocked in 5% nonfat milk for 2 h at room temperature and subsequently incubated with primary antibodies overnight at 4 °C. The primary antibodies targeted the following proteins: NLRP3 (1:500), ɑ- tubulin (1:1000), GAPDH (1:3000) (Cell Signaling Technology, USA), ASC (1:200), procaspase-1, caspase-1 (1:500), p-PKC α (1:500) (Santa Cruz, USA), IL-1β, pro-IL-1β (1:1000), PKC α (1:1000), and occludin (1:2000) (Abcam, USA). Then, the membranes were incubated with secondary antibodies (1:5000) for 2 h at room temperature, and ECL Super Signal reagent (Millipore, USA) was used to detect protein bands. Image J software was used to measure the relative band density of different proteins on the scanned membrane [1 (link), 8 (link)].