Af4005

After the treatments, the cells were lysed on ice for 20 min with a mixture of 100 μl RIPA buffer (Fdbio science, Hangzhou, China), 1 μl protease inhibitor (Fdbio science) and 1 μl phosphatase inhibitor (Fdbio science) and centrifuged at 14,000 g for 25 min. Protein concentrations were determined using a BCA Total Protein Assay Kit (CWBIO, China). Protein samples (40 μg/lane) were separated on SDS-PAGE gels (8% or 12%, Epizyme, China) and electrotransferred to PVDF membranes. The blots were incubated with the following primary antibodies: anti-NLRP3 (1:500, A12694, ABclonal), anti-GSDMD (1:500, 39754, CST), anti-p20 (1:500, AF4005, Affinity), anti-CHOP (1:500, ab11419, Abcam), anti-GRP78 (1:500, ab21685, Abcam), anti-COL2A (1:500, ab34712, Abcam), anti-Aggrecan (1:500, ab36861, Abcam), anti-MMP3 (1:500, ab52915, Abcam), anti-MMP13 (1:500, ab39012, Abcam), anti-ADAMTS5 (1:1000, ab41037, Abcam), and anti-SREBP1 (1:1000, ab28481, Abcam). The membranes were washed and incubated with the HRP-conjugated secondary antibodies (1:8000, ABclonal, China) for 1 h. The bands were developed with chemiluminescence reagents and imaged by a myECL imager (Syngene G:BOX ChemiXT4, United Kingdom). The experiment was repeated in triplicate, and the blots were quantified by ImageJ software. An antibody against β-actin (AC026, 1:1000; ABclonal) served as an endogenous control.

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.

The inferior lobe of the lung tissues was homogenized in RIPA lysis buffer (Thermo Fisher Scientific, Inc.). After that, 30 μg protein was separated in 10% SDS-PAGE. Proteins were then transferred to PVDF membranes (Millipore). Then, the PVDF membranes were blocked with 5% non-fat dry milk for 1 h at room temperature and then incubated with primary antibodies against p-P65 (1 : 1000, AF2006, Affinity); P65 (1 : 1000, AF5006, Affinity); p-IKBα (1 : 1000, AF2002, Affinity); IKBα (1 : 1000, AF5002, Affinity); NLRP3 (1 : 1000, DF7438, Affinity), ASC (1 : 2000, sc-514414, Santa Cruz Biotechnology, INC), Caspase-1 p20 (1 : 2000, AF4005, Affinity), Pro-GSDMD (1 : 1000, AF4012, Affinity), GSDMD p30 (1 : 1000, DF12275, Affinity), IL-18 (1 : 1000, DF6252, Affinity), IL-1β (1 : 1000, AF5103, Affinity), and β-actin (1 : 5000, AF7018, Affinity) at 4°C overnight. Then, the membranes were then incubated with the appropriate secondary antibodies at room temperature for 2 h. β-actin was selected as the loading control. Finally, the protein bands were caught by using a Chemiluminescence image analysis system (Tanon, Shanghai, China) and analyzed with the Image J software (National Institutes of Health, USA).