Bicinchoninic acid protein assay kit

The renal cortex was lysed in radioimmunoprecipitation assay (RIPA) lysis buffer with phenylmethanesulfonyl fluoride (PMSF), loading buffer, and phosphatase on ice, and protein concentration was determined by the bicinchoninic acid protein assay kit (Biosharp, China). Proteins from renal cortex lysates were denatured in boiling water for 10 minutes, loaded to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto nitrocellulose membranes. The membranes were blocked for 1 hour with 5% bovine serum albumin (BSA) in Tris-buffer saline containing 0.1% Tween 20 (TBST) at room temperature. The membrane was incubated overnight at 4°C with a primary antibody, then washed with TBST for five times and incubated with horseradish peroxidase-conjugated secondary antibodies (Beyotime, China) at room temperature for 1 hour and developed with an enhanced chemiluminescence agent. The membranes were placed on the ChemiDoc Touch Imaging System (Bio-Rad Laboratories, USA) to image a protein band, and ImageJ (Adobe Corp., USA) was used to determine band intensity. Protein expression was quantified as the ratio of a specific band to β-actin.

HUVECs and BMECs were lysed using radioimmunoprecipitation assay buffer (Biosharp, Hefei, China). To determine protein concentration, a Bicinchoninic Acid Protein Assay Kit (Biosharp) was utilized. Equivalent protein lysates were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Then they were then subjected to western blot analysis. The primary antibodies against FOXO1 (2880S), cleaved caspase-3 (9661S), and β-actin (3700S) were purchased from Cell Signaling Technology (Danvers, MA, USA). The primary antibodies anti-cyclin D1 (60186-1-1), anti-cyclin E1 (11554-1-AP), anti-Bim (22037-1-AP), anti-Bcl2 (60178-1-Ig), anti-Bax (60267-1-Ig), anti-matrix metalloproteinase (MMP)2 (66366-1-Ig), anti-MMP9 (N-terminal, 10375-2-AP), and anti-vascular endothelial growth factor A (VEGFA; 66828-1-Ig) were obtained from Proteintech. In addition, horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG H&L (ZJ2020-M and ZJ2020-R, respectively; Bioworld) was applied as the secondary antibody. The protein bands were visualized via an electrochemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed using Image Lab 5.0 software (Bio-Rad, Hercules, CA, USA).

The tissues or cultured cells were lysed by radioimmunoprecipitation assay (RIPA; Santa Cruz Biotechnology, Santa Cruz, California, USA) supplemented with a protease inhibitor and phosphatase inhibitor cocktail (Roche, Shanghai, China). Then, a bicinchoninic acid protein assay kit (Biosharp, Shanghai, China) was used to detect the protein concentration. For electrophoresis, 20 μg of the protein sample was loaded on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred into polyvinylidene fluoride membrane (PVDF; Millipore, CA, USA) after electrophoresis. Then, membranes were blocked in Tris-buffered saline Tween 20 (TBST) containing 5% skimmed milk for 1 h. These membranes were incubated with primary antibody overnight at 4°C (SRPK1, ab58002, Abcam; Phospho-ERK (Thr202/Tyr204), #4370, Cell Signaling Technology). After washed with TBST for 3 times, PVDF membranes were further incubated with goat secondary antibody IgG-horseradish peroxidase at room temperature for 1 h. Finally, X-ray film (Kodak, NY, USA) was used to analyze the optical density value of target bands. β-actin was served as an internal standard for normalization. Results of densitometric analysis were measured by ImageJ software.