Anti col1a1

Human TGFβ1 (Solarbio, No. P00121) was suspended as recommended by the manufacturer, at 10 μg/mL stock concentration. Aliquots were kept frozen at −20°C until used. TRPC3 specific inhibitor Pyr3 was purchased from Sigma-Aldrich (St. Louis, MO).
Western blot assays were conducted as previously described [19 (link), 20 (link)]. The primary antibodies included anti-TRPC3 from Alomone Labs (Jerusalem, Israel); anti-TGFβ1 and anti-αSMA from Abcam (Cambridge, UK); anti-fibronectin, anti-NOX4, anti-phosphorylated Smad2/3 (pSmad2/3), anti-Smad2/3, and anti-GAPDH from Santa Cruz Biotechnology (Dallas, TX); anti-Col1a1 from Cell Signaling Technology; and antiphosphorylated pyruvate dehydrogenase E1a subunit (PDHE1a) (p-PDHE1α) from Merck-Millipore (Darmstadt, Germany).

Keratocytes, seeded onto glass coverslips and treated as previously described for 72 h, were fixed with 3.7% buffered paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS and washed with PBS. Slides were then blocked with 1% bovine serum albumin in PBS for 1 h at room temperature, and finally incubated overnight at 4 °C with the following primary antibodies: anti-CD34 (1:50 dilution; catalog no. ab81289; Abcam), anti-α-SMA (1:100 dilution; catalog no. ab7817; Abcam), anti-COL1A1 (1:300 dilution; catalog no. #39952; Cell Signaling Technology), or anti-podoplanin (1:200 dilution; catalog no. ab10288; Abcam). The day after, slides were incubated for 45 min at room temperature in the dark with Alexa Fluor-488-conjugated or Rhodamine Red-X-conjugated secondary antibodies (1:200 dilution; Invitrogen). Irrelevant isotype-matched and concentration-matched mouse and rabbit IgG (Sigma-Aldrich) were used as negative controls. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Immunostained cells were examined with a Leica DM4000 B microscope (Leica Microsystems) and fluorescence images were captured with a Leica DFC310 FX 1.4-megapixel digital color camera equipped with the Leica software application suite LAS V3.8 (Leica Microsystems). The assay was performed in duplicate for each of the five cell lines.

Proteins from kidney tissues and cultured cells were extracted with RIPA lysis buffer (Beyotime). Protease and phosphatase inhibitor cocktails were added for the extraction of phosphorylated proteins. The total protein concentration was measured using a BCA protein assay kit (Beyotime). Proteins (40–80 μg) were separated using SDS-PAGE (10%) and transferred to a PVDF membrane. The membranes were blocked with 5% BSA for 1.5 h and incubated with primary antibodies overnight at 4°C, followed by secondary antibodies (goat anti-rabbit, 1:3000; anti-mouse, 1:5000; Servicebio, Wuhan, China) for 1.5 h. The following primary antibodies were used: anti-α-SMA (1:3000, Proteintech, Wuhan, China), anti-Col1a1 (1:1000, Cell Signaling Technology), anti-collagen type III alpha 1 chain (anti-Col3a1, 1:1000, Abclonal), anti-Cpt1a (1:1000, Abclonal), anti-Acox1 (1:1000, Abclonal), anti-phosphorylation-AMP-activated protein kinase (anti-p-AMPK, 1:1000, Abclonal), and anti-glyceraldehyde-phosphate dehydrogenase (anti-GAPDH, 1:50000, Abclonal). Images were visualized using a Gene Gnome XRQ system (Syngene, Cambridge, UK).