Anti vhl antibody

Tissue sections were deparaffinized twice by xylene and then hydrated. Hydrogen peroxide (0.6%) was used to eliminate endogenous peroxidase activity. The sections were blocked with goat serum in Tris-buffered saline for 30 min. Sections were then incubated with anti-VHL antibody (Abcam, 1:100) and anti-Twist1 antibody (Abcam, 1:500) overnight at 4 °C. Secondary antibody was then applied and incubated at 37 °C for 1 hour. Sections were developed with diaminobenzidine and stopped with water. Photographs of representative fields were captured using a Leica CCD camera DFC420 connected to a Leica DMIRE2 microscope (Leica Microsystems Imaging Solutions, Cambridge, UK). Quantification of immunoreactivity was performed on digitally captured color images saved as TIFF files and analyzed using Image-Pro plus 6.0 (Media Cybernetics, Rockville, Maryland, USA). These methods were performed in accordance with the approved guidelines and regulations from the Research Ethics Committee of Huashan Hospital, Fudan University.

Anti-FLAG antibody was purchased from eBioscience (Cat#14-6681-82), anti-panCK antibody
from Biogenex (Cat#AM273-5 M), anti-VHL antibody from Abcam (Cat#ab140989), and
anti-CK8/18 from Novus (Cat#NBP2-44929). Murine ccRCC cell line RENCA and human ccRCC cell
line ACHN, prostate cancer cell lines CWR22v1 and C4-2 were purchased from the American
Type Culture Collection (ATCC) and maintained in RPMI-1640, supplemented with 10% fetal
bovine serum and penicillin/Streptomycin at a working concentration of 100 U/mL. Murine
prostate cancer cells Myc-CaP were purchased from ATCC and maintained in DMEM,
supplemented with 10% fetal bovine serum and penicillin/Streptomycin at a working
concentration of 100 U/mL. Human bladder cancer cell lines T24 and HT-1376 were kind gifts
from Dr. Arnold I. Chin and Dr. Hanwei Zhang at UCLA and maintained in the same condition
as RENCA cells.
Lentiviral plasmid encoding mStrawberry and EGFP, together with flag tag or HA, and
plasmid encoding firefly luciferase were constructed based on pSicoR (Addgene, #11579),
and lentivirus was packaged as mentioned previously in the report²⁶ (link).