Anti ck18

Cells were prepared and lysed in ice-cold RIPA buffer (cat. no. 89900; Thermo Fisher Scientific, Inc.) containing freshly added Halt Phosphatase Inhibitor Cocktail and Protease Inhibitor Cocktail, and protein concentrations were determined using the Bradford protein assay (Bio-Rad Laboratories, Inc.). Samples of 20 µg proteins were separated via SDS-PAGE on a 12% gel (Bio-Rad Laboratories, Inc.) and proteins were immunoblotted onto nitrocellulose membranes. The membranes were blocked in 5% non-fat milk for 1 h at room temperature and probed with primary antibodies [anti-β-actin (1:1,000; cat. no. 60008-1-Ig; ProteinTech Group, Inc.), anti-Vim (1:1,000; cat. no. ab92547; Abcam) or anti-CK18 (1:1,000; cat. no. ab133263; Abcam)] at 4˚C overnight, followed by HRP-conjugated secondary antibodies [HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (cat. no. SA00001-1; 1:2,500; ProteinTech Group, Inc.) and HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (cat. no. SA00001-2; 1:2,500; ProteinTech Group, Inc.)] for 2 h at room temperature. The blots were visualized using Immobilon Western Chemiluminescent HRP Substrate (cat. no. WBKLS0500; MilliporeSigma), and images were captured using the ECL Tanon 5500 system (Tanon Science and Technology Co., Ltd.). Protein expression was normalized to the expression levels of β-actin.

HK-2 cells or renal tissue samples were rinsed with ice-cold PBS and lysed using RIPA lysis buffer (Beyotime, Shanghai, China). The lysates were centrifuged at 12,000×g at 4°C for 10 min, and the supernatants were collected to measure protein concentration by BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of proteins (30 μg) were separated by 12% SDS-PAGE and then electrophoretically transferred to a PVDF membrane (Beyotime, Shanghai, China). The membranes were blocked with 5% skim milk in TBST and then incubated with primary antibodies (anti-CK-18: Abcam, 1:1000; anti-α-SMA: Abcam, 1:1000; anti-MMP9: Abcam, 1:1000; anti-p-Smad2: Abcam, 1:1000; anti-p-Smad3: Abcam, 1:1000; anti-Collagen III: Abcam, 1:1000; anti-Fibronectin: Abcam, 1:1000; anti-β-actin: Abcam, 1:1000; Cambridge, MA, USA) at 4°C overnight. Then, membranes were incubated with secondary antibodies (Goat Anti-Rabbit IgG: Abcam, 1:5000, Cambridge, MA, USA) at room temperature for 1 h. The bands of proteins were visualized using a chemiluminescent detection system (BeyoECL Plus, Beyotime, Shanghai, China). Finally, band densities were examined using IPP Image-Pro Plus software. β-actin was used as the internal control.