Ab7817

After dewaxing with xylene, kidney paraffin sections were immersed in gradient alcohol and water before staining, then soaked in 3% H₂O₂ to remove endogenous peroxidase. All sections were heated in sodium citrate solution for 15 min in 98°C for antigen retrieval. Then the tissue specimens were circled with an immunohistochemical pen, added 5% bovine serum albumin (BSA) solution, and blocked at 37°C for 30 min, incubated with the primary antibodies including fibronectin (1:500, abcam, ab268021, United Kingdom, Cambridge), alpha smooth muscle actin (1:300, abcam, ab7817, United Kingdom Cambridge), collegen-I (1:500, abcam, ab270993, United Kingdom, Cambridge) solution 200 μL at 4°C overnight. After washing off, these tissues were incubated with secondary antibody solution at 37°C for 40 min, colored by 3,3′-diaminobenzidine (DAB) under a microscope and counterstained with hematoxylin (Cat. No. C0105S, Beyotime Biotechnology, Shanghai, China). The immunohistochemistry kits were purchased from Boster Biotechnology (Fujian, China). DAB color reagent kits were purchased from Maixin Biotechnology (Fujian, China).

Whole-cell proteins were extracted and subjected to standard western blot analysis as described previously [41 (link)]. Antibodies against MYOCD (SAB4200539, Sigma, Massachusetts, USA), ACTA2(ab7817, Abcam, Massachusetts, USA), SM22α (ab14106, Abcam, Massachusetts, USA), PCNA (sc-56, Santa Cruz, California, United States), OPN (sc21742, Santa Cruz, California, United States), LC3 (NB100-2220, Novus Biologicals, Colorado, USA), P62 (610833,BD Transduction Laboratories, USA), Beclin1 (ab207612, Abcam, Massachusetts, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245, Abcam, Massachusetts, USA) were used to probe for the target proteins. Secondary antibodies, including IRDye^®800CW anti-rabbit secondary antibody and IRDye^®680 anti-mouse secondary antibody, were purchased from Li-COR (Lincoln, Michigan, USA). Signals were detected by an Odyssey™ Infrared Imaging System (LI-COR, Michigan, USA), and the bands were quantified by Image-Pro Plus 5.1 software (MEDIA CYBERNETICS, Maryland, USA).

For immunohistochemical analyses, the sections were exposed overnight at 4°C to antibodies against E-cadherin (rabbit monoclonal; 1 : 200; ab76319; Abcam), COL I (rabbit monoclonal; 1 : 200; ab270993; Abcam, Cambridge, UK), MMP-9 (rabbit monoclonal; 1 : 200; ab76003; Abcam), TIMP-1 (rabbit polyclonal; 1 : 200; Abcam), α-SMA (mouse monoclonal; 1 : 200; ab7817; Abcam), or fibronectin (rabbit monoclonal; 1 : 200; ab199056; Abcam), followed by incubation with secondary horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1 : 500; 115-035-003; Jackson ImmunoResearch, Ely, UK).

Total protein was extracted for 30 min with 50 mL radioimmunoprecipitation assay buffer containing 0.5 mL protease inhibitors. Total protein concentrations were quantified by the bicinchoninic acid protein assay. Primary antibodies against CK19 (1:1000; A19040, ABclonal, United States), TGFβR1 (1:1000; ab31013, Abcam, United States) and α-SMA (1:1000; ab7817, Abcam, United States) were used for the analysis. Chemiluminescence measurements and semiquantitative values were obtained by a ChemiDocXRS+Imaging system and Image Gauge V3.12. Protein levels were quantified relative to β-actin.

All cell culture serum and media were purchased from Thermo Fisher Scientific, while cell culture supplements were purchased from Sigma. Rat anti-CD31 (553369) was purchased from Pharmingen while antibodies against alpha SMA (ab7817), phosphor-ErK (ab50011) and ErK1/2 (ab17942) were from Abcam. The antibodies against phospho-Akt (sc-7985R), Akt1 (sc-1619) and GAPDH (sc-25778) were from Santa CruZ Biotech. The antibody against VEGF (SAB1402390) was from Sigma. All secondary antibodies were from Dakocytomation. All microRNA reagents were purchased from Thermo Fisher Scientific. PDGF, insulin, holo-transferrin, SU5416, LY294002, DMSO, DAPI and Giemsa were purchased from Sigma.

Whole cells from ex vivo experiments were prepared by 1× cell lysis buffer (9803, Cell Signaling Technologies) with protease inhibitors (04693159001, Roche Molecular Biochemicals, USA). Protein concentrations were determined by using a BCA protein assay. Proteins were separated by SDS–PAGE, transferred to polyvinylidene fluoride membranes, and incubated overnight at 4 °C with antibodies diluted with blocking liquid including anti-PAD4 (ab2148, Abcam, 1:800), anti-CD68 (NB100-683, Novus, 1:500), anti-α-SMA (ab7817, Abcam, 1:1000), anti-H3CIT (ab5103, Abcam, 1:500), anti-GAPDH (60004-1-Ig, Proteintech, 1:5000), anti-MYD88 (SC-74532, Santa Cruz, 1:600), anti-TLR4 (SC-10741, Santa Cruz, 1:500), anti-pSTAT3 (9145, Cell Signaling Technology, 1:1000), anti-Stat3 (9139, Cell Signaling Technology, 1:1000), anti-SOCS1 (ab9870, Abcam,1:800), and anti-STING (ab288157, Abcam, 1:1000). Then incubated with secondary antibody for 1 h and bends were visualized using chemiluminescence (TANON, China) and viewed under Amersham Imager 600 system (GE Healthcare, USA). Uncropped scans of the immunoblots are supplied in the Source Data file.