Pb0589

Proteins were extracted from cells, and their concentration was measured using a bicinchoninic acid protein assay kit (Beyotime, China). Total protein was separated by 8% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The following primary antibodies were used: osteopontin (OPN, 1 : 1000, PB0589, Boster), focal adhesion kinase (FAK, 1 : 500, BM4303, Boster), p-FAK (1 : 500, BM4426, Boster), Ras (1 : 400, BM4281, Boster), mitogen-activated protein kinase (MAPK, 1 : 1000, BM4439, Boster), p-MAPK (1 : 2000, P00176, Boster), phosphatidylinositol 3-kinase (PI3K, 1 : 1000, BM5187, Boster), p-PI3K (1 : 1000, ab182651, Abcam), and GAPDH (1 : 1000, PAB36264, Bioswamp). After three washes with phosphate-buffered saline/Tween 20, the membranes were incubated with horseradish peroxidase-conjugated secondary goat anti-rabbit IgG (1 : 20000, PAB160011, Bioswamp). Protein bands were visualized by enhanced chemiluminescence color detection (Tanon-5200, TANON) and analyzed using AlphaEase FC gel image analysis software.

The kidney tissues were cut up and lysed in RIPA Lysis Buffer with protease inhibitor phenylmethanesulfonyl fluoride (PMSF) and phospho-proteinase inhibitors (Beyotime Biotechnology, Shanghai, China). The protein concentration was measured using the bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, Shanghai, China). Proteins were separated and isolated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis with 5% and 12% for 120 V, 2 h and then transferred onto polyvinylidene fluoride (PVDF) membranes for 200 mA, 70 min (MCP-1, IL-6) and 200 ma, 90 min (BMP2, OPN). The PVDF membranes were blocked with 5% bovine serum albumin for 2 h. The membranes were incubated with primary antibody against BMP2 (proteintech group, 66,383–1-Ig, China, 1:1500), OPN (Boster, PB0589, China, 1:1000), IL-6 (Boster, BA4399, China, 1:1000), MCP-1 (proteintech, 66,272–1-Ig, China, 1:1500), β-actin (proteintech, 66,009–1-Ig, China, 1:4000) at 4 °C overnight. Then they were incubated with secondary antibody at room temperature for 2 h, the proteins were visualized using enhanced developer (Boster, China). The gray values of these proteins were analyzed with image-pro plus. All the WB quantifications were used three independent Western blot results.