Primary antibodies against α sma

The tissue was harvested at indicated time points and lysed in a lysis buffer (0.5% Nonidet P-40, 10 mM Tris, pH 7.4, 150 mM Nacl, 1 mM EDTA, 1 mM Na₃VO₄) with a protease inhibitor (1 mM PMSF). BCA assay was used to quantify protein level. Cell lysates (30 μg protein) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Immobilon; Millipore, Bedford, MA). For immunoblotting, primary antibodies against α-SMA, TGF-β, α-tubulin and β-actin (1:1000) (Abcam, Cambridge, UK) and IgG-HRP secondary antibody (1:2000) were used. Chemiluminsescent signals were acquired using Storm 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Densitometric analysis was performed using Image J 5.0 software. Four biological replicates were performed.

Kidney tissues were fixed in 4 % formalin and embedded in paraffin. Paraffin sections (4-μm) were stained with Masson’s trichrome and Sirius Red. Renal fibrosis was assessed and quantified by imaging analysis (ImageJ software; Bethesda, MD, USA). Briefly, 6–8 corticomedullary junction viewing fields were selected from appropriate areas for each kidney examined. Fibrosis was expressed as the percentage of the total area. A total of 4–5 images were evaluated per kidney, and mean values were calculated. Immunohistochemical staining was performed on kidney by using routine protocols [59 (link)]. Briefly, sections were blocked with 5 % BSA for 1 h at room temperature and incubated at 4° C overnight with primary antibodies against α-SMA, LTβR, Sphk1 (All diluted by 1:150; Abcam, Cambridge, MA, USA), and TNFSF14, HVEM (All diluted by 1:150; Santa Cruz, Dallas, TX, USA). These slides were then stained with a horseradish peroxidase-conjugated secondary antibody (1:800; Beyotime, Shanghai, China). The results were analyzed using the DAB assay kit (ZSGB-BIO, Beijing, China). The slides were visualized by microscopy (Olympus BX51, Japan).

Thioacetamide (TAA) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in normal saline (0.9% sodium chloride). Primary antibodies against α-SMA, collagen-I, Smad4, and B-cell lymphoma 2 (Bcl-2)-associated X protein (BAX) were purchased from Abcam (Cambridge, UK). ER-α, Smad7, Bcl-2, p53, and β-actin were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Vimentin, p-Smad2/3, Smad2/3, cleaved caspase 3, p-PI3K, p-Akt, phosphatase and tensin homolog (PTEN), and p-PTEN were purchased from Cell Signaling Technology (Danvers, MA, USA). Assay kits used to measure aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transferase (γ-GGT), malondialdehyde (MDA), and catalase (CAT) were purchased from Abcam (Cambridge, UK). Total glutathione (GSH) assay kits were purchased from Enzo Life Sciences (New York, NY, USA). SOD assay kits were purchased from Cayman Chemical (Ann Arbor, MI, USA). Hydroxyproline assay kits were purchased from Cell Biolabs, Inc. (San Diego, CA, USA). All other chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA).