Ecl luminescence kit

Proteins were isolated from various cultured NRK52E cells and NRK49F cells. As previously described, the protein levels were determined using Western blot analysis.19 (link) The primary antibodies used were as follows: anti-Claudin-2 (1:500, A14085, ABclonal, China), anti-PCNA (1:500, 10205-1-AP; Proteintech, China), anti-α-SMA (1:100, 14395-1-AP, Proteintech, China), anti-Collage I (1:500, 14695-1-AP,Proteintech, China), CTGF (1:500, A11067,ABclonal, China),anti-GAPDH (1:5000, 10494-1-AP, Proteintech, China), anti-β-actin (1:5000, 20536-1-AP; Proteintech, China). Finally, the ECL luminescence kit (E412-02, Vazyme, China) was used for luminescence. Moreover, quantification was performed by measuring the signal intensity using the Image J software (National Institutes of Health, Bethesda, MD, United States).

The total protein in HK-2 cell was collected by RIPA lysis method and the protein concentration was detected by using BCA kit (PC0020, Solarbio, China). Then the proteins were separated using 12% SDS-PAGE (G2037, Servicebio, China) electrophoresis and transferred to PVDF membrane via the electro-blotting at 210 mA for 75 minutes. After taking out the PVDF membrane, wash it three times with TBST for 15 minutes each time. A 5% skimmed milk powder was added and sealed for 2 h. Primary antibody (1:1,000) was put in and cultivated in a refrigerator at 4 ℃ overnight. The next day, TBST was used for washing three times to remove primary antibody, HRP labeled secondary antibody (1:5,000) was added. After incubation for 2 h, TBST was used for washing three times, with 15 min each time. Finally, ECL luminescence kit (E412-02, Vazyme, China) was used for luminescence and the results of WB analysis were performed by using the software ImageJ. The data were extracted from three independent biological experiments.