A14394

Adjacent normal tissues and ccRCC tissues were fixed in formalin, dehydrated, embedded, and then incubated with primary rabbit TRIM2 polyclonal antibody (1:100; A14394; ABclonal Biotech Co., Ltd., Wuhan, China) at 4°C overnight. The sections were washed three times with PBS and, then, incubated with goat antirabbit secondary antibody (1:200; GB23303; Servicebio, Inc., Woburn, MA, USA) for 2 hours at room temperature.

Tissues and cells were pyrolysised in protein lysis system containing RIPA (Wuhan Boster Biological Technology, Ltd.), protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA), and phenylmethylsulfonyl fluoride (PMSF) (Wuhan Boster Biological Technology, Ltd.). Concentrations of protein were measured using the bicinchoninic acid kit (Beyotime Institute of Biotechnology, Haimen, China) at 562 nm. A total of 30 µg of proteins were subjected to SDS-PAGE and, then, separated and transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA) for 90 minutes. After transferred to PVDF membranes, the proteins were blocked in PBS with 5% nonfat milk for 1 hour and then incubated with antibodies against TRIM2 (1:1,000; A14394; ABclonal Biotech Co., Ltd.) and GAPDH (1:2,000; BM3876; Wuhan Boster Biological Technology, Ltd.) at 4°C overnight. The membranes were washed and incubated with secondary antibodies (1:5,000; BA1020; Wuhan Boster Biological Technology, Ltd.) on the next day at room temperature for 2 hours. Finally, the membranes were washed and, then, detected by ChemiDoc-XRS+ (Bio-Rad Laboratories Inc., Hercules, CA, USA).