Anti tgf β1

A piece of kidney tissue (100 mg) was homogenized in 1 mL radio immunoprecipitation assay lysis buffer and 10 μL phenylmethanesulfonyl fluoride (100 mmol/L). After centrifugation at 12,000× g for 10 minutes at 4°C, the supernatant was collected. The total protein concentrations were determined by using a bicinchoninic acid protein assay kit. Protein samples (50 μg) were run on sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking with 5% skim milk for 1 hour, membranes were incubated with primary antibodies (anti-CD31, Santa Cruz Biotechnology, 1:1,000; anti-α-SMA, Wuhanboshide, 1:1,000; anti-TGF-β1, Bioworld Technology, Nanjing, People’s Republic of China, 1:1,000) overnight at 4°C. Immunoreactive bands were detected by the use of chemiluminescent horseradish peroxidase substrate, and scans were obtained using the Bio-Rad gel image analysis system (Bio-Rad Laboratories Inc., Hercules, CA, USA) and processed with Image-Pro Plus (Media Cybernetics, Inc.).

Paraffin-embedded sections of mouse liver tissues were used to detect the expression of proteins, according to the manufacturer’s protocol. Tissue sections were incubated at 4°C overnight with the following rabbit anti-mouse primary antibodies, anti-TGFβ-1 (1: 400) (Bioworld Technology Inc., St. Louis Park, MN, USA), anti-SMAD3 (1: 400) (Bioworld Technology Inc., St. Louis Park, MN, USA), anti-SMAD7 (1: 400) (Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-HIF-1α (1: 200) (Santa Cruz Biotechnology Inc., Dallas, TX, USA), and anti-VEGF (1: 200) (Santa Cruz Biotechnology Inc., Dallas, TX, USA). The tissue sections were then incubated with biotinylated IgG at room temperature for 1 h and incubated with a goat anti-rabbit secondary antibody for 15 minutes. Tissue sections were washed three times with PBS, incubated with streptavidin for 15 minutes, rinsed with PBS, and incubated in the 3,3-diaminobenzidine (DAB) chromogen, and the reaction was stopped with tap water. The tissue sections were counterstained with hematoxylin and then coverslipped. Photomicrographs were taken by senior pathologists. The proteins were semi-quantified with Image J analysis software (National Institutes of Health, USA).