Rabbit anti c6orf15

Total protein was extracted from three infected CRC cell lines (HCT116, RKO, and MC38) using RIPA lysis buffer supplemented with 1% PMSF. Total protein was separated using a 10% sodium dodecyl sulfate‒polyacrylamide gel and then transferred to a PVDF membrane. The membranes were blocked with 5% skim milk for 2 h and then incubated with rabbit anti-C6orf15 (Proteintech, USA; 1:1000 dilution), rabbit anti-β-catenin (Proteintech, USA; 1:1000 dilution), rabbit anti-ZEB1 (Abmart, Shanghai; 1:1000 dilution), rabbit anti-E-cadherin (Abmart, Shanghai; 1:1000 dilution), rabbit anti-N-cadherin (Abmart, Shanghai; 1:1000 dilution), rabbit anti-Vimentin (Proteintech, USA; 1:1000 dilution), rabbit anti-ZO-1 (Abmart, Shanghai; 1:1000 dilution), mouse anti-GAPDH (Abmart, Shanghai; 1:3000 dilution), rabbit anti-CPT1A (Proteintech, USA; 1:1000 dilution), and rabbit anti-LaminB1 (Proteintech, USA; 1:1000 dilution). The membranes were then incubated with secondary antibodies for 2 h. We assessed the protein blotting results using an enhanced chemiluminescence (ECL) assay kit (Thermo Fisher Scientific, Rockford, IL, USA), and each experiment was performed three times.

Tissue sections of liver metastases and primary foci of colon cancer from the same patient were placed in xylene, anhydrous ethanol, 95%, 85%, and 75% alcohol in series and in water for deparaffinization and then placed in citric acid antigen repair solution. Next, the sections were incubated in 4% BSA for 30 min before they were incubated with a primary antibody [rabbit anti-C6orf15 (Proteintech, USA; 1:100 dilution)] at 4 °C overnight. The sections were then incubated with a fluorescent secondary antibody (Proteintech, USA). Nuclei were visualized using DAPI (Sigma, St. Louis, MO, USA), and the sections were analysed using an EVOS fully automated imager (Invitrogen, USA).