P1081

For IB analysis, cell lysis buffer (P0013, Beyotime) supplemented with PMSF (ST506, Beyotime) and phosphatase inhibitor cocktail was used to lyse the cells (P1081, Beyotime). Proteins were isolated using polyacrylamide gel electrophoresis with sodium dodecyl sulfate and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk and the indicated primary antibodies at 4 °C overnight. After being washed three times with TBST buffer, the cells were incubated with the HRP-conjugated secondary antibodies (SA00001-1, SA00001-2; Proteintech) and detected by using chemiluminescence (34580, Thermo Fisher).
For IP analysis, cell lysis buffer (P0013, Beyotime) containing PMSF and phosphatase inhibitor cocktail was used to lyse the cells for IP analysis (P1081, Beyotime). Cell lysates were first precleared with protein A/G agarose, and then were incubated with the indicated primary antibodies at 4 °C overnight. The next day, 40 μl of protein A/G-agarose beads were added and incubated for 4 h at 4 °C. After washing three times, the mixture was resuspended. After boiling and centrifugation to pellet the agarose beads, supernatants were subjected to IB analysis.

Western blots were performed as previously described [25 (link)]. Total protein was extracted from kidney tissue in RIPA lysis buffer (25 mM Tris-HCl, 25 mM NaCl, 0.5 mM EDTA, 1% Triton X-100, and 0.1% SDS) with 1% PMSF protease inhibitors (P1005, Beyotime Biotechnology, China) and phosphatase inhibitors (P1081, Beyotime Biotechnology, China) added. Equal amounts of protein were separated by 10-15% SDS-PAGE and then transferred to PVDF membranes (IPVH00010, Millipore, Germany). After blocking with 5% nonfat milk for 3 h, the membranes were incubated with the primary antibodies including TGF-β1, α-SMA, Nrf2, HO-1, NQO1, ASC, caspase-1 (ab215715, ab32575, ab137550, ab13248, ab80588, ab175449, ab1872, Abcam, UK), p-Smad2, Smad2, p-Smad3, Smad3, E-cadherin, NLRP3 ((#18338, #5339, #9520, #9523, #3195, #15101, Cell Signaling Technology, USA), and cleaved caspase-1 (AF4005, Affinity Biosciences, USA) at 4°C overnight. Then, the membranes were washed 3 times with TBST and incubated with the secondary antibodies for 1 h at room temperature. After washing with TBST for a further three times, the protein bands were visualized by enhanced chemiluminescence (32134, Thermo, USA) solution and imaged with Automated Imaging System (Gene Gnome5, Synoptics Ltd, UK). GAPDH or β-actin was assumed to be similarly abundant in all samples and was used as a loading control.

A mixed solution of radio immunoprecipitation assay lysis buffer (RIPA, P0013C, Beyotime, Shanghai, China), phenylmethanesulfonyl fluoride (PMSF, ST506, Beyotime, Shanghai, China), and phosphatase inhibitor (P1081, Beyotime, Shanghai, China) was added to the hippocampal tissue to extract the total protein. Then, the hippocampal tissue was thoroughly ground and centrifuged at 13,000 rpm for 5 min at 4 °C, and the centrifuged supernatant was collected and assayed for the protein concentration using the BCA protein assay kit (BL521A, Biosharp, Guangzhou, China). Each mouse took 10 μL of hippocampal tissue samples for ELISA detection. The concentrations of proinflammatory cytokines in the hippocampus, including TNF-α (FEK0527, BOSTER, Wuhan, China) and IL-1β (EK0394, BOSTER, Wuhan, China), were detected by a commercial ELISA kit. The experimental procedures were carried out in strict accordance with the manufacturer’s instructions. The concentrations of TNF-α and IL-1β were presented as pg/mg protein.