Anti sgol1

Total protein was isolated using radioimmunoprecipitation assay (RIPA) lysis buffer (Merck Millipore, Waltham, MA, USA) and quantified using the BCA kit (Solarbio Co., Ltd., Beijing, China) from cell lines or HCC tissues. After being subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), samples were transferred onto polyvinylidene difluoride (PVDF) membranes (0.45 μm; Merck Millipore), blocked with 5% skimmed milk in Tris buffer and 0. 1% Tween-20 for 2 h, followed by incubation at 4 °Covernight with anti-SGOL1 (Proteintech, Wuhan, China), Cyclin D1 (Proteintech), Cyclin E1 (Wuhan, China), GAPDH (1:1000, Servicebio Co., Ltd., Wuhan, China), CDK7 (1:1000, Proteintech), CDK4 (1:1000, Servicebio Co., Ltd.), and P27 (1:1000, Proteintech) antibodies. Secondary antibodies were incubated for 2 h after membranes were thrice washed with TBST. Bands were displayed using the ultrasensitive ECL chemiluminescence kit (Boster Co., Ltd., Wuhan, China), and protein expression level was visualized by an imaging system (Bio-Rad Laboratories, Hercules, CA, USA).

The proteins were extracted from ccRCC cells and HK-2 cells with RIPA buffer (Beyotime Institute of Biotechnology, China) supplemented with 1% protease and phosphatase. The total protein concentration was subsequently detected with a BCA kit (Thermo, Germany). The proteins (20 μg) were subjected to SDS–PAGE and transferred onto polyvinylidene fluoride membranes (FVDF, 0.45 μm; Merck Millipore). After being blocked with 5% skim milk for 1 h and incubated with anti-SGOL1 (Proteintech, 16,977–1-AP), anti-P21 (Proteintech, 10,355–1-AP), anti-cyclin D1 (Proteintech, 26,939–1-AP), anti-CDK2 (Proteintech, 10,122–1-AP), anti-β-actin (Proteintech, 20,536–1-AP), anti-MMP2 (CST, 40,994), anti-MMP9 (CST, 13,667), anti-cyclin E1 (CST, 20,808), anti-E-cadherin (CST, 3195), and anti-N-cadherin (CST, 13,116) antibodies at 4°C overnight, the PVDF bands were incubated with secondary antibodies and detected by enhanced ECL chemiluminescent assay and visualized by Bio-Rad software after washing with TBST three times.