Horseradish peroxidase labeled goat anti rabbit antibody

Cells in 15 ml culture were collected by centrifugation. Cell pellets were re-suspended in 1 ml 10 mM Tris–HCl buffer (pH 8.0). The resulting cell suspensions were sonicated to disrupt Sulfolobus cells. Cell debris was removed by centrifugation 13000 rpm at 4°C for 30 min, yielding cellular extracts for further analysis. Protein concentrations of the samples were determined using Coomassie Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Similar amounts of protein (ca. 10 μg) were taken from the prepared cell extracts and loaded on a 12% polyacrylamide gel for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, fractionated proteins were transferred from the polyacrylamide gel onto a nitrocellulose blotting membrane (GE Healthcare, Waukesha, WI, USA), using the Semi-Dry Electrophoretic Transfer Cell system (Bio-Rad, Hercules, CA, USA) and used for immunoblotting. Briefly, the membrane was incubated in 5% skim milk blocking agent for 1 h, and then incubated with individual primary rabbit antibodies (against Orc1-2 or PCNA3 proteins) and finally with the horseradish peroxidase-labeled goat anti-rabbit antibody (Beyotime, Beijing, China) as described previously (31 ). Protein bands were visualized using the ECL western blot substrate (Thermo Scientific, Waltham, MA, USA) and recorded by exposure to an X-ray film.

The sections of xenograft tumor tissues (n=9) were stained with H&E, and the histologic features were described using a DP26 optical microscope (Olympus Optical Co., Ltd., Kyoto, Japan). Xenograft tumor tissues (n=9) were homogenized, and the supernatant was collected. The proteins were separated by 12% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% non-fat milk, incubated with rabbit anti-human CAIX monoclonal antibody (1:1,000; Abcam) at 4°C overnight and horseradish peroxidase-labeled goat anti-rabbit antibody (1:1,000; Beyotime Institute of Biotechnology) for 1 hour. After incubation with enhanced chemiluminescence reagents, the bands were analyzed using Image Lab™ software version 5.2.1 (Bio-Rad Laboratories Inc., Hercules, CA, USA).