Anti tgf β1

Cells were lysed in buffer (20 mM Tris-HCl pH 8, 150 mM NaCl) containing a protease inhibitor. Protein concentrations were determined using a BCA Protein Assay Kit. A total of 20–30 μg of proteins were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. The membrane was blocked with 5% (w/v) non-fat milk in Tris-buffered saline with 20% TWEEN-20 (TBS-T). Membranes were then incubated in primary antibodies overnight: anti-TGF-β1 (Santa Cruz Biotechnology), Anti-α-SMA (Sigma-Aldrich), Anti-collagen I (Santa Cruz Biotechnology), Anti-fibronectin (Abcam), Anti-iNOS (Abcam), Anti-IL-10 (Santa Cruz Biotechnology), Anti-p-Smad2 (Cell Signaling Technology), Anti-Smad2 (Santa Cruz Biotechnology), Anti-p-Smad3 (Cell Signaling Technology), Anti-Smad3 (Santa Cruz Biotechnology), Anti-Smad7 (Santa Cruz Biotechnology), and Anti-p-NF-κB, Anti-NF-κB, Anti-p-IκBα, Anti-IκBα (Cell Signaling Technology). After incubation, anti-rabbit IgG and anti-goat IgG (Santa Cruz Biotechnology) were used to detect proteins. The membranes were visualized using an enhanced chemiluminescence detection (ECL) kit (Amersham Pharmacia Biotech, Piscataway, NJ, United States). Densitometric analysis was performed using ImageJ software¹.

Protein expression was analyzed using Western blotting as previously described (Yu et al. 2019 (link); Gong, Yan, et al. 2019 (link)). For Western blot analysis, primary antibodies against IL-6 (mouse monoclonal antibody, ab9324) and nephrin (rabbit monoclonal antibody, ab216341) were purchased from Abcam (UK), Anti-TGF-β1 (mouse monoclonal antibody, sc-130348) was purchased from Santa Cruz (USA), anti-podocin (rabbit polyclonal antibody, TA351459) was purchased from Origene (USA), anti-caspase-3 (rabbit polyclonal antibody, 9662S) was purchased from CST (USA), and anti-ALB (rabbit polyclonal antibody, 16475-1-AP) was purchased from Proteintech (USA). Goat anti-mouse IgG-HRP (ZB2305) and goat anti-rabbit (ZB2301) IgG-HRP secondary antibodies were purchased from ZSGB-Bio (Beijing, China). The targeted proteins were visualized with the Super Signal West Femto Chemiluminescent Substrate (Thermo Scientific Pierce), and the intensities of the visualized bands were analyzed using Quantity One software (Bio-Rad). β-Actin (mouse monoclonal antibody, TA-09, from ZSGB-Bio in Beijing, China) was used as an internal control. The data are expressed as the ratio to β-Actin.

Paraffin-embedded kidneys were sectioned at 4 μm. The sections were air-dried, dewaxed, and rehydrated in graded ethanol and phosphate buffered saline (PBS). Endogenous peroxidase activity was blocked using 3% hydrogen peroxide and the sections were blocked with 10% goat serum. The primary antibodies were anti-TGF-β1(1:200; Boster Bioengineering Co., Wuhan, China), anti-TGF-β1 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Smad2 (1:200; #ab40855; Abcam, Cambridge, MA, USA), anti-Smad7 (1:200; #BA1499, Boster Bioengineering Co., Wuhan, China), and anti-Smad3 (1:1000; #ab40854, Abcam, Cambridge, MA, USA). All sections were subsequently incubated with the respective secondary antibodies at room temperature for 1 h. Peroxidase-labeled polymer and diaminobenzidine (DAB) were used to visualize staining. Three to five sections of each specimen were used for immunohistochemistry. A qualitative analysis was first performed; according to the location of the specific protein in the control group (e.g., the cortical part), the same location was analyzed in the treatment groups. Semiquantitative analysis was then conducted using eight randomly selected nonoverlapping fields (×400) from each section. The quantitative analysis was conducted using Image Pro Plus 6.0.