Bs 1303r

Total protein from the kidney tissue and NRK-52E cells was obtained with a protein extraction kit (Solarbio, Beijing, China) as directed by the manufacturer. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed to separate proteins (50 μg/well). When bromophenol blue ran to the bottom of the gel, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. Blocking was carried out with 5% skim milk. The membranes were then washed with TBST three times for 10 min and subjected to incubation with the following antibodies overnight: β-catenin (1:500, bs-1165R, Bioss), Sfrp1 (1:500, bs-1303R, Bioss), p-GSK3β^ser9 (1:500, sc-373800, Santa Cruz), E-cadherin (1:500, bs-10009R, Bioss), and α-SMA (1:500, 55135-1-AP, Proteintech), collagen IV (col-IV) (1:1000, SAB4200500, Sigma), β-actin (1:4000, Lot:181620, Pumei), and GSK3β (1:500, sc-8257, Santa Cruz). The next day, the membranes underwent three TBST washes of 10 min. Secondary antibodies diluted with 1% skim milk were added to the membranes for 1 h at room temperature. Band intensities were assessed by Image Lab software.

For immunohistochemistry, a two-step immunoassay kit (ZSBIO, Beijing, China) was used for streptavidin-peroxidase (SP) detection. For immunofluorescence, NRK-52E cells were grown in 12-well plates, stimulated with normal or high glucose, and transfected with miR-27a inhibitor, miR-27a inhibitor negative control, si-sfrp1, and combined miR-27a inhibitor and si-sfrp1. After treatment for 48 h, the cells underwent fixation (4% formalin at room temperature, 20 min), permeabilization (0.1-0.3% Triton X-100, 10 min), and blocking (3% BSA at 37°C, 30 min). Next, sfrp1 antibody (1:50; bs-1303R, Bioss), β-catenin antibody (1:50; bs-1165R, Bioss), E-cadherin antibody (1:50; bs-10009R, Bioss), α-SMA antibody (1:50; 55135-1-ap, Proteintech), and col-IV antibody (1:50; SAB4200500, Sigma) were added for overnight incubation at 4°C. The cells were then rinsed with ice-cold PBS, and the corresponding fluorescent secondary antibodies were added for incubation (37°C, 1 h). DAPI staining was performed for 5 min, and the cells were observed under an immunofluorescence microscope (Leica, DM4000B, Germany).