Collagen 1

Cardiomyocytes and mouse heart tissue were lysed as previously reported 9 (link). After quantities by BCA assay, equal total proteins were separated in SDS-PAGE gels (10-12%). After transferred onto PVDF membranes, protein bands were blocked with 5% BSA. After washing with TBST, membranes were blotted with primary antibodies at 4°C overnight as follows: Bax (Abclonal), BCl₂ (Affbiotech), Caspase-3 (Abclonal), Collagen I (Bioworld), Phospho-NF-κB p65 (Ser536) (Cell Signaling Technology,), NF-κB (Cell Signaling Technology), GAPDH (Bioword), and ß-actin (Bioword). Quantifications of Western Blots was done using Image J.

Tissue sections were blocked with 3% BSA for 30 min at 25 °C. Sections were incubated overnight at 4 °C with antibodies against integrin αVβ5 (1:100, Santa Cruz Biotechnology, Japan). For GC and TC staining, sections were fixed in 4% paraformaldehyde (Servicebio, China) for 30 min at 25 °C and then permeabilized with 0.3% Triton X-100 (Beyotime, China). After washing with PBS three times, the cells were blocked with 3% BSA for 30 min at 25 °C. Cells were incubated with antibodies against IRE1α (1:100, Proteintech), apoptosis-associated speck-like protein containing a CARD (ASC, 1:100, AdipoGen Life Science, USA), NLRP3 (1:100, CST, USA), a-smooth muscle actin (α-SMA, 1:100, Abcam, UK) and collagen I (1:100, Bioworld Technology, China) overnight at 4 °C. After washing with PBS three times, tissue sections and cells were incubated at 25 °C for 2 h with fluorescent secondary antibodies (Beyotime). Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Beyotime) at a dilution of 1:2000 for 30 min and photographed using an Olympus laser scanning confocal microscope (FV3000, Japan).

Western blotting was performed for the detection of TGFβ1, smad2/3, phosphorylated Smad2/3, a-SMA, collagen I and collagen III. Total proteins were extracted from bladder tissues and analyzed with bicinchoninic acid protein concentration assay kit (Beyotime Institutes of Biotechnology, Shanghai, China). Proteins were isolated from the samples by SDS-PAGE electrophoresis and then transferred onto nitrocellulose membrane. After blocking with 5% nonfat milk or 2% bovine serum albumin (BSA), the membranes were incubated at 4°C overnight with a rabbit anti-rat polyclonal TGFβ1 (Santa Cruz Technology, 1:800dilution), smad2/3 antibody (Santa Cruz Technology, 1:1000dilution), p-smad2 antibody (Bioword Technology, 1:800dilution), p-smad3 (Cell signaling,1:1000dilution), α-SMA(Cell signaling,1:1000dilution), collagen I (Bioworld Technology, 1:800dilution) and collagen III (Bioworld Technology,1:800dilution). The second antibody was a goat anti-rabbit IgG (1:10000dilution, Li-COR, USA), which was diluted in TBST. Signals were scanned and visualized by Odyssey Infrared Imaging System (LiCor Inc., Lincoln, NE). Density of western strap was quantified by Image J software, with the gray value representing the amount of expressed target protein. Relative protein levels were calculated by comparing the gray value of target protein to the amount of β-actin protein.