Interleukin 1β il 1β

The detection of protein expression was performed by Western blotting as described previously [18 (link)]. Briefly, kidney samples were collected and lysed in the RIPA buffer (Millipore Technology, Billerica, MA, USA). After boiling, 20 μg samples were loaded into the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8–12%), transferred to the PVDF membrane (Millipore Technology, Billerica, MA, USA), and then blocked with 5% bovine serum albumin (BSA). Subsequently, the PVDF membrane was incubated with primary antibodies overnight at 4 °C, including fibronectin (BD Biosciences, San Jose, CA, USA), α-SMA (Sigma-Aldrich, St. Louis, MO, USA), E-cadherin, phosphorylated NFκB-p65 (p-p65), the total form of NFκB-p65, cleaved PARP, cleaved caspase 3, α-tubulin, cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), and CTGF (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and then incubated in the secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Finally, the protein expression levels of the PVDF membrane were detected by the enhanced chemiluminescence kit (Millipore Technology, Billerica, MA, USA) in a digital photo-image system.

Sodium taurocholate (Na-TC) and sodium pentobarbital were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lentiviral vectors encoding ATG7 (Lv-ATG7) or CAMKII (Lv-CAMKII) and lentivirus with scrambled sh-ATG7 (Lv-sh-ATG7) or sh-CAMKII (Lv-sh-CAMKII) were purchased from GeneChem (Shanghai, China). MiR-30b-5p mimic and mimic negative control were purchased from RiboBio (Guangzhou, China). The primary antibodies used for Western blot were microtubule-associated protein 1 light chain 3 (LC3), p62, ATG7 and high mobility group protein B1 (HMGB1) purchased from Cell Signaling Technology (Danvers, MA, USA), tumor necrosis factor α (TNF-α), β-actin and interleukin-1β (IL-1β) purchased from Santa Cruz Biotechnology (Dallas, Texas, USA), CAMKII purchased from Abcam (Shanghai, China), and lysosome-associated membrane protein-2 (LAMP-2) purchased from Thermo Fisher Scientific (Rockford, IL, USA).

Proteins (40 μg per lane) were separated with SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). After being blocked with 5% skim milk in TBST for 1 h, the membranes were incubated with primary antibody against Dynamin-related protein 1 (DRP1; 1:1,000; Abcam), Mitofusin 2 (MFN2; 1:1,000; Abcam), NLRP3 (1:1,000; Cell Signaling Technology, Danvers, MA, USA), caspase-1 (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), GSDMD (1:500; Abcam), interleukin-1β (IL-1β; 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-18 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), synapsin 1 (1:2,500; Millipore, Billerica, MA, USA), PSD-95 (1:1,500; Abcam), voltage-dependent anion channel (VDAC; 1:1,000; Cell Signaling Technology, Danvers, MA, USA), or GAPDH (1:1,000; Cell Signaling Technology, Danvers, MA, USA). After three washes, the membranes were treated with species-specific peroxidase-conjugated secondary antibodies for 1 h at room temperature. Bands were visualized by enhanced chemiluminescence and quantified with Image Quant Software (Syngene).