Anti gsdmd

Rabbit polyclonal antibodies against caspase-1, NLRP3 and ASC were made in house and were described previously56 (link)57 (link)58 (link). T7̇Tag monoclonal antibody HRP conjugate (Catalogue No. 69048) was from Novagen. Monoclonal anti-β-actin (Catalogue No. A-5316) was from Sigma Aldrich. Anti-DFNA5 (Catalogue No. sc-79233), anti-caspase-3 (Catalogue No. sc-7148), anti-GSDMD (Catalogue No. sc-393656) and anti-Bax (Catalogue No. sc-930) were from Santa Cruz. Anti- Na,K-ATPase polyclonal antibody (Catalogue No. 3010) was from Cell Signaling Technology. zVAD-fmk (Catalogue No. A1902) was obtained from ApexBio. GSK'872 (Cat No. AOB4887) was obtained from Aobious. CytoTox96 LDH-release kit (Cat No. G1780) was from Promega. TALON metal affinity resin (Catalogue No. 635502) and In-Fusion HD Cloning Plus (Catalogue No. 638910) were obtained from Takara Clontech. All antibodies were used at 1/1,000–1/2,000 dilutions for western blot analyses.

For immunoblot analysis, both the cells adherent to the dishes and the cells floating in the medium were collected by low-speed centrifugation. The collected cells were lysed in lysis buffer [0.32 M sucrose, 10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 50 mM NaF, 2 mM Na₃VO₄ and a protease inhibitor cocktail (Roche, Mannheim, Germany)], and the protein concentrations of the cell lysates were determined by the method of Bradford (1976) (link). Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to poly vinylidene fluoride (PVDF) membranes, and probed with following antibodies: anti-caspase-1 (ab179515, abcam, Cambridge, MA), anti-cleaved caspase-3 (#9661, Cell Signaling Technology, Beverly, CA), anti-GSDMD (sc-393656, Santa Cruz Biotechnology, Santa Cruz, CA), anti-DFNA5/GSDME (ab215191, abcam), and anti-actin (A2066, Sigma-Aldrich, St. Louis, MO). Then, antigens were visualized by use of peroxidase-conjugated anti-IgG antibodies (Promega, Madison, WI) and a Western Lightning Chemiluminescence Reagent Plus Kit (Perkin Elmer Life Science, Boston, MA).

Kidneys samples were fixed in 10% neutral formalin and paraffin-embedded sections were stained Periodic acid-Schiff (PAS) and Sirius red. Samples were scored by an experienced pathologist in a blinded fashion for signs of acute tubular injury. Fibrosis was quantitated in Sirius red-stained sections using MRI fibrosis tool and Color Deconvolution plugin for “ImageJ [https://github.com/MontpellierRessourcesImagerie/imagej_macros_and_scripts/wiki/MRI_Fibrosis_Tool]”. Ten similarly oriented photos in the same part of the kidney at the same magnification were studied per biological sample. Color deconvolution and simple auto-thresholding were applied and the fibrosed area of the selection was measured and compared to the area of the selection on the input image.
Immunofluorescence staining was performed after deparaffinization and antigen retrieval using citrate buffer pH 6.0 with a pressure cooker (PickCell Laboratories, Agoura Hills, CA). Antibodies were diluted in blocking buffer (TBS 0.1% Tween 20, 0.05% Triton X-100, 5% bovine serum albumin) as follows: anti-GSDMD (1:100, Santa Cruz, #393656), anti-SLC34A1 (1:100, Novus, #NBP2-13328), anti-CD11B (1:50, BD, #555386), AF555-conjugated donkey anti-rabbit (1:1000, LifeSciences, #A31572), AF488-conjugated goat anti-mouse (1:1000, LifeSciences, #A11029).

The FDB or soleus muscles from mice were homogenized by sonication in cold RIPA lysis buffer, containing 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM BAPTA, 20 mM Tris-HCl, pH 7.5, 1% Triton X-100, and Protease Inhibitor Cocktail (Roche Diagnostics, Germany), as described previously [20 (link)]. The samples were loaded onto a 12% SDS-polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA). The primary antibodies used were: anti-NLRP3 (1:500) from R&D Systems (MAB7578, Minneapolis, MN, USA), anti-IL-1β (1:500; SC-32294), anti-caspase-1 (1:500; SC-56036), anti-ASC (1:1000; SC514414), and anti-GSDMD (1:500; SC-393581) from Santa Cruz Biotechnology (Dallas, TX, USA), and anti-GADPH (1:20000) from Sigma-Aldrich (G9545; St. Louis, MO, USA). After washing, the membranes were incubated for 1.5 h with secondary anti-rabbit, anti-rat, or anti-mouse antibodies, as appropriate (Sigma-Aldrich, St. Louis, MO, USA). Western blotting detection reagents (LumiFlash™ Infinity Chemiluminescent Substrate, Taipei, Taiwan) were used following the manufacturer’s instructions, and chemiluminescence was detected using a ChemiDoc Imaging System from Bio-Rad (Hercules, CA, USA). The intensity of the bands was quantified by densitometry, with the use of ImageJ software version 1.44p (NIH, Bethesda, MD, USA).

The following antibodies were used for immunostaining: anti-AIF1 (1:500 dilution) from ThermoFisher (Waltham, MA), anti-GSDMD (1:500 dilution) from Santa Cruz (Dallas, TX), and anti-Ki67 (1:100 dilution) from Abcam (Cambridge, MA). TUNEL assay and all qRT-PCR primers were purchased from ThermoFisher.