Nbp1 30054

In brief, slides were fixed with 4% PFA for 15 min at RT and permeabilized in ice cold menthol for 6 min at −20 °C. Heat-mediated antigen retrieval with a 0.01 M citric acid (pH 6.0) was performed for 5 min in a microwave. After 4% BBBSA (4% IgG-free BSA in PBS; Jackson, 001-000-162) blocking, the sections were further blocked with unconjugated AffiniPure Fab Fragment (1:100 in PBS; Jackson, 115-007-003) for 30 min. The biotin-conjugated anti-mouse IgG (1:500 in 4% BBBSA, Jackson, 115-065-205) and Cy3-Streptavidin (1:1250 in 4% BBBSA, Jackson, 016-160-084) were used as secondary antibodies. Primary antibodies and dilutions were used as following: mouse anti-PAX7 (1:50, DSHB), mouse anti-MyoD (1:100, Dako, M3512), mouse anti-eMyHC (1:300, Leica, NCL-MHC-d), rabbit anti-Collagen1 (1:200; Novus, NBP1-30054), and rabbit anti-laminin (1:800, Sigma-Aldrich, L9393). Masson’s trichrome staining was performed according to the manufacturer’s instructions (ScyTek Laboratories, Logan, UT). All fluorescent images were captured with a fluorescence microscope (Leica, DM 6000B). Measurements of Collagen 1 and collagen positive area were conducted by in house ImageCount software written in MATLAB (R2014b) language.

Total protein was extracted from the whole kidney or cultured cells using RIPA buffer (0.05 M Tris, 0.15 M NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, pH7.4) with PMSF, protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche). The concentration of the protein was determined using the BCA protein assay kit (Beyotime). For Western blot, equal amount of protein (20–50 μg per lane) was loaded in a 7.5% or 10% SDS-PAGE mini-gel and transferred to a PVDF membrane (Millipore). The membrane was blocked with 5% BSA in PBST buffer (100 mM PBS, pH 7.5, 0.1% Tween-20) and then incubated in primary antibody diluted with blocking buffer overnight at 4 °C. The primary antibodies included: anti-TNC (IBL, 10337), anti-collagen Iα (Novus, NBP1-30054), anti-αSMA (Sigma, A2066), anti-STAT3 (CST, 12640), anti-phospho-STAT3 (Tyr 705, Αbcam, ab76315), anti-EGFR (abcam, ab52894), anti-phospho-EGFR (TYR1068, CST, 2234), anti-GAPDH (CST, 5174) and anti-β-actin (Proteintech, 66009-1-Ig) antibody. Membranes were then incubated with appropriate secondary antibodies and subjected to chemiluminescence detection using ECL Reagent (Millipore, WBKLS0500).