Ab3848 1

Cells (1×10⁷) under 80% confluence or IVD tissues (0.1 g) were lysed in 1 mL ice-cold radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher; #89901) containing the protease inhibitor (Abcam; #ab142778). Equal amounts (30 µg) of proteins were loaded into the wells of SDS-PAGE gel and separated by electrophoresis. Proteins were transferred onto the PVDF (polyvinylidene fluoride) membrane (Sigma-Aldrich; #IPSN07852) and blocked with 5% fat-free milk for one hour at room temperature. The membranes were then incubated with primary antibodies, including anti-BMPR1a (Abcam; #ab264043), anti-BMPR1b (Abcam; #ab175385), anti-BMPR2 (Abcam; #ab96826), anti-Smad1/5/8 (Sigma-Aldrich; #SAB2702532), anti-pSmad1/5/8 (Sigma-Aldrich; #AB3848-I), anti-Smad4 (Sigma-Aldrich; #HPA019154), anti-Puma (Abcam; #ab9645), anti-Apaf-1 (Abcam; #ab233786), anti-CASP9 (Abcam; #ab184786), anti-CASP3 (Thermo Fisher; #MA1-16843), anti-HDAC1 (Abcam; #ab7028), and anti-β-actin (Sigma-Aldrich; #A2066). After incubation at 4 °C overnight, membranes were washed 5 times with a PBS buffer containing 0.1% Tween-20 (Sigma-Aldrich; #P9416) and then probed with secondary antibodies (Abcam; #ab6721 and #ab6728). Protein signals were recorded by the Bio-Rad Gel Imaging System (Bio-Rad, Shanghai, China; #1708265).

The fibroblasts were seeded with 150.000 cells/well and treated for 60 min with Activin A or 90 min with BMP4 after serum starvation overnight. Whole-cell lysates were prepared by lysing cells in NuPAGE^® LDS Sample Buffer with 10% NuPAGE^® reducing agent. Proteins were separated in NuPAGE 4–12% BT gels using the XCell SureLock™ system and were subsequently transferred by using the iBlot Dry Blotting system (Invitrogen). Nitrocellulose membranes were blocked in the Odyssey blocking buffer (Westburg). Immunoblotting was performed in Odyssey blocking buffer with 0.1% Triton X-100.
Primary antibodies against phospho-SMAD3 (cat#52903, Abcam), phospho-SMAD1/5/9 (AB3848-I, Sigma-Aldrich), and Actin (Cat#ab14128, Abcam) were used for overnight incubation. Secondary antibody incubation was carried out for 1 h with the IRDye 800 CW goat anti-rabbit IgG and IRDye 680 CW goat anti-mouse (LI-COR Biosciences). Fluorescence was visualized by the Odyssey system equipped with the Odyssey version 4 software (LI-COR Biosciences).

Immunohistochemical and immunofluorescent studies were performed as described previously (Jaimes et al. 2007 (link); Lan et al. 2018 (link)). Primary antibodies were rabbit anti-Grem1 (Novus Biologicals, NBP1-31150, 1:100), rabbit anti-Nephrin (Abcam, Cambridge, MA, ab58968, 1:100), rabbit anti-P-Smad2/3 (Abcam, ab272332, 1:100), rabbit anti-P-Smad1/5/8 (Sigma-Aldrich, Burlington, MA, AB3848-I, 1:100). Periodic acid-Schiff (PAS) staining was conducted by specialists at the Pathology Department, Long Island-Jewish Hospital (Queens, NY), following standard staining protocols. Masson trichrome staining was performed by specialists at the Pathology Department, Shanghai Gefan Biotechnology Co., Ltd (Shanghai, China), following standard staining protocols.