Anti gapdh

Western blotting analysis was performed as previously reported. Briefly, cells were lysed in immunoprecipitation lysis buffer (Beyotime Biotechnology, Shanghai, China) with 1 mM PMSF on ice for 30 min. Protein concentrations were measured by the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA). Equal amounts of protein were separated by SDS-PAGE and transferred onto 0.22 μm nitrocellulose membranes (Millipore, Cork, Ireland). The membranes were incubated with anti-SPP1 (Abcam, ab214050), anti-GAPDH (ABclonal Biotechnology, Hubei, China, A19056), anti-CD44 (Abcam, ab189524), anti-SMAD2/3 (Abcam, ab202445), anti-pSMAD2/3 (Abcam, ab254407), anti-STAT3 (Abcam, ab68153), and anti-pSTAT3 (Abcam, ab76315) overnight at 4 °C, and then incubated with IRDye 800 goat anti-rabbit antibody (LI-COR Biosciences, Lincoln, USA) for 1 h at room temperature. After washing off the unbound antibodies, the labeled bands were scanned by Odyssey® CLx Infrared Imaging System (LI-COR Biosciences, MA, USA).

The protein samples were extracted by RIPA lysis buffer (Beijing Applygen Technologies Co., Ltd., Beijing, China) supplemented with protease and phosphatase inhibitors (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and denatured in boiling water for 10 min. The denatured proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, USA). The membranes were blocked in PBST (0.05% Tween-20) solution containing 5% nonfat milk for 1 h to remove nonspecific binding. Following overnight incubation with the primary antibodies anti-RELA (1:1000, ABclonal, Wuhan, China) or anti-GAPDH (1:500,000, ABclonal, Wuhan, China) at 4 °C, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000, Shanghai Sangon Biotech, Shanghai, China) at room temperature for 1 h. An enhanced chemiluminescence (ECL) system was applied to visualize protein bands, and ImageJ software was used to quantify protein expression levels.

The primary antibodies include anti-β-actin (Santa Cruz), anti-GAPDH (ABclonal), anti-SREBP2 (Abcam, for western blotting), anti-SREBP2 (Santa Cruz sc-271616 for immunohistochemistry), anti-mTOR (Cell Signaling Technology), anti-phospho-mTOR-ser2481 (Cell Signaling Technology), anti-phospho-mTOR-ser2448 (Cell Signaling Technology), anti-MET (Cell Signaling Technology), anti-phospho-MET (Cell Signaling Technology), anti-AKT (Cell Signaling Technology), anti-Phospho-Akt (Ser473) (Cell Signaling Technology), anti-Phospho-Akt (Thr308) (Cell Signaling Technology), anti-p70 S6 Kinase (Cell Signaling Technology), anti-Phospho-p70 S6 Kinase (Cell Signaling Technology). HGF neutralizing antibody was purchased from R&D Systems, Inc.

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).

Total proteins were extracted from H9C2 cells or cardiac tissues using RIPA buffer (Solarbio, Beijing, China, R0020) supplemented with protease and phosphatase inhibitors (Sangon Biotech, Shanghai, China, C600387, and C500017, respectively). The following antibodies were used in this study: anti-Vps4a (Sigma, SAB4200215), anti-LC3 (Sigma, L8918), anti-p62 (CST, 5114), anti-GAPDH (Abclonal, Wuhan, China, AC002), anti-p-AKT (CST, Boston, MA, USA, 4060), anti-AKT (CST, 4685), anti-Myh7 (Santa, Dallas, TX, USA, sc-53090), anti-HRP conjugated goat anti-rabbit IgG polyclonal Ab (HuaBio, Hangzhou, China, HA1001), and anti-HRP conjugated goat anti-mouse IgG polyclonal Ab (HuaBio, HA1006). The immunoreactions were visualized with Super Signal West Pico PLUS (Thermo Fisher Scientific, Waltham, MA, USA, 34577) and imaged by Azure C400 Imaging Systems.

The content of TNF-α, collagen type I, and collagen type III in infarcted myocardium was assessed by Western blot. The protein concentrations were measured with BCA protein Assay Kit (Beyotime Biotechnology, China). Equal amounts of protein (20 μg) were added into each lane of a 10% SDS-PAGE (New Cell & Molecular Biotech, China) and separated. Then, the protein was transferred onto PVDF membranes (Millipore, United States). And the membranes were blocked by Protein Free Rapid Blocking Buffer (EpiZyme, China) for two h and incubated with primary antibodies (anti- TNF-α, 1:1000, Abclonal, China; anti-collagen type I, 1:1000, Proteintech, United States; anti-collagen type III, 1:5000, Proteintech, United States; anti-GAPDH, 1:1000, Abclonal, China) at 4 ℃ for 12 h. After incubation with HRP-coupled secondary antibody (goat anti-rabbit or rabbit anti-mouse IgG, 1:10,000, Boster, China), the membranes were treated with chemiluminescence (Solarbio, China) for 3 min and visualized on a Visionwork system.