Nbp1 31150

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.

Western blotting was performed as described in our previous publications (Lan et al. 2013 (link), 2016 (link); Wen et al. 2019 (link)). The acquired images were analyzed using the NIH image program in the public domain. Primary antibodies used were rabbit anti-Grem1 (Novus Biologicals, NBP1-31150, 1:1000), rabbit anti-P-Smad2/3 (Abcam, ab63399, 1:1000), rabbit anti-P-Smad1/5/8 (Millipore Sigma, AB3848-I, 1:1000), and mouse anti-actin (Santa Cruz Biotechnology, sc-8432, 1:3000). For protein expression quantification, films were scanned using a Canon Scan 9950F scanner.