Ab2413

Western blot analyses were performed as described previously [37 (link)]. The protein concentrations of cell or tissue lysates were measured using the Lowry assay. Afterward, 40 μg protein samples were separated using 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE, according to the molecular weight of the proteins of interest) and then electroblotted onto a nitrocellulose membrane. The membranes were blocked with BlockPRO blocking buffer (BP01-1L; Visual Protein, Taipei, Taiwan), immunoblotted with specific primary antibodies against THBS2 (1:1000, A8561; ABclonal, Woburn, MA, USA), HSP90 (1:500, sc-101494; Santa Cruz Biotechnology), transferrin (1:1000, GTX112729; Gene Tex, Irvine, CA, USA), CD 81 (1:500, GTX101766; Gene Tex), α-SMA (1:1000, bsm-52396R; Bioss Antibodies Inc, Woburn, MA, USA), β-catenin (1:1000, GTX632676; Gene Tex), Fibronetin (1:500, ab2413; Abcam, Cambridge, MA, USA), Vimentin (1:1000, SC-7557; Santa Cruz Biotechnology), and β-actin (1:1000, NB600-501; Novus Biologicals, Centennial, CO, USA), and then detected using horseradish peroxidase-conjugated secondary antibodies. The signals were visualized by chemiluminescence using an enhanced detection kit (ECL, TU-ECL03, TOOLS, Taipei, Taiwan).

The primary antibodies against fibronectin (FN, ab2413), type IV collagen (Col-IV, ab6586), quinolinate phosphoribosyltransferase (QPRT, ab171944, for cell Western blotting), nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1, ab45652, for kidney tissue Western blotting and immunofluorescence staining) and nicotinamide riboside kinase 1 (NRK1, ab169548, for cell Western blotting) were purchased from Abcam (Cambridge, MA, USA). Antibodies of anti-vimentin (#5741), anti-α-Tubulin (#3873) and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (#7074) were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against α-smooth muscle actin (α-SMA, A5228), β-actin (A5441) and QPRT (SAB1410425, for kidney tissue Western blotting and immunofluorescence staining) were from Sigma-Aldrich (St Louis, MO, USA). The anti-NMNAT1 (sc-271557, for cell Western blotting) and anti-NRK1 (sc-398852, for kidney tissue Western blotting and immunofluorescence staining) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against nicotinic acid phosphoribosyltransferase 1 (NAPRT1, 13549-1-AP), nicotinamide phosphoribosyltransferase (NAMPT, 11776-1-AP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 60004-1-IG) and HRP-conjugated anti-mouse IgG (SA00001-1) were purchased from Proteintech Group (Wuhan, China).

For the characterization of primary fibroblasts isolated from patients, cells were seeded in glass coverslips. The following day, cells were fixed with 4% paraformaldehyde for 10 min at room temperature, permeabilized with 0.125% Triton X‐100 for 10 min and blocked with 4% BSA for 1 h. Primary antibodies against Fibronectin (ab2413, 1 : 100; Abcam, Cambridge, UK) and Vimentin (sc‐6260, 1 : 50; Santa Cruz, CA, USA) were incubated overnight in a humidified chamber at 4 ºC. Coverslips were washed three times with PBS containing 0.1% Tween‐20 and incubated for 1 h at room temperature with the respective secondary antibody (1 : 400; Abcam). Finally, coverslips were again washed and mounted in ProLong Diamond Antifade mounting media containing 4′,6‐diamidino‐2‐phenylindole (DAPI; Thermo Fisher Scientific, Waltham, MA, USA). Samples were imaged with Zeiss Cell Observer inverted microscope and processed using imagej (NIH).

Cells were lysed with ice-cold RIPA buffer and centrifuged at 13,000 g for 15 min at 4 °C. 20 ug of protein lysate (concentration determined by bicinchoninic acid assay) was run on a 10% SDS-PAGE gel at 120 V for 1.5 h and then transferred to nitrocellulose filter membranes (Millipore, Bedford, MA). After blocking with 5% BSA at room temperature (RT) for 1 h, the membranes were incubated with the following primary antibodies: α-SMA (#19245, 1:1000, Cell Signaling Technology, Danvers, MA), fibronectin (ab2413, 1:1000, Abcam, Cambridge, UK), arginase-1 (Arg1) (16001-1-AP, 1: 5000, Proteintech, Wuhan, China), ornithine δ-aminotransferase (OAT) (A6235, 1:1000, ABclonal Technology, Wuhan, China), PYCR1 (13108-1-AP, 1:4000, Proteintech), proline dehydrogenase (oxidase) 1 (PRODH) (22980-1-AP, 1:2000, Proteintech) and β-actin (20536-1-AP, 1:5000, Proteintech) at 4 °C overnight. The next day, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG(H + L) (SA00001-2, 1:10000, Proteintech) for 1 h at room temperature and then washed three times for 10 min with tris-buffered saline containing 0.1% Tween 20. Quantitative analysis was performed on the immunoreactive bands using ImageJ software.

Hearts were harvested, fixed with 4% paraformaldehyde and then paraffin‐embedded. After that, 5‐mm sections were made. Masson staining were employed to assess cardiac fibrosis along with collagen deposition by a Masson stain kit. The whole‐heart fibrosis (fibrosis area/whole heart area) and left ventricular CVF (collagen area/total observed area in multiple random ×20 magnification visual fields) were computed as the ratio of fibrosis area to the overall or left ventricular assessed area quantified with the ImageJ software (V.1.50, Bethesda, USA). Soaking of the sections in 3% H₂O₂ was done to stop the endogenous peroxidase activity. Subsequently, the sections were inoculated overnight with anti‐NLRP3 antibody (Novus, NBP2‐12446, USA; 1:200), anti‐α‐SMA (Abcam, ab124964, USA; 1:200) and anti‐Fibronectin (Abcam, ab2413, USA; 1:200) at 4°C for immunohistochemistry. We randomly selected 5 vision fields (×20) for every section with a microscope (Olympus, Tokyo, Japan).

Protein was extracted from cardiac tissue with 1× RIPA buffer containing 0.1% SDS. Protein (20 µg) was subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes. Primary antibodies for β-MHC (1:1000, #AF7533, Beyotime, Shanghai, China), fibronectin (1:1000, #ab2413, Abcam), and GAPDH (1:10,000, #2118S, CST). The appropriate secondary antibodies conjugated to horseradish peroxidase (HRP) were from Santa Cruz Biotechnology (1:10,000). Membranes were visualized with SuperSignal West Pico Substrate (Pierce Biotechnologies, Rockford, IL, USA) or Immobilon Western HRP Substrate (Millipore, Billerica, MA, USA). Quantification was performed by using ImageJ software.