Anti satb1

Western blotting was performed to determine SATB1 expression in human ccRCC tissues and cell lines. Briefly, total protein was extracted using RIPA Buffer (Sigma, St Louis, MO, USA), and protein concentrations were quantified by BCA Protein Assay Kit (Boster Ltd., Wuhan, China). Equal amounts of harvested protein samples were resolved on a 10% SDS–PAGE and transferred to PVDF membranes (Millipore Corporation, Billerica, MA, USA). The blotted membranes were blocked with 5% BSA in TBST for 2 h at room temperature, and incubated with the indicated primary antibodies overnight at 4°C. The rabbit polyclonal anti-SATB1 (Abcam Inc., Cambridge, CA, USA) was used at the dilution of 1∶1000, while GAPDH (Boster Ltd., Wuhan, China) was used as a loading control. The membranes were washed and then incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Boster Ltd., Wuhan, China) for another 2 h at room temperature. Immunoblots were visualized by enhanced chemiluminescence using Supper Signal West Pico Trail Kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer's instructions.

Immunofluorescence staining with the antibody to SATB1 was performed to determine the expression and distribution of SATB1 in human RCC cell lines. Cells cultured on a 24-well plate were fixed in 4% paraformaldehyde for 20 min at 37°C, and then washed twice with PBS for 10 minutes. After permeabilized with 0.3% Triton for 15 minutes, cells were blocked with 5% BSA for 30 min. Subsequently, cells were incubated with a rabbit polyclonal anti-SATB1 (1∶200; Abcam Inc., Cambridge, MA, USA) overnight at 4°C, followed by visualization with a goat anti-rabbit IgG-TRITC (1∶100; Jackson, West Grove, PA, USA) for 1 hour at room temperature in the dark. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) for 5 min. Images were evaluated with a Nikon-A1Si confocal microscope (Nikon Corporation, Tokyo, Japan).