The S2-647 monoclonal antibody recognizes the Von Hippel-Lindau (VHL) protein. The VHL protein is expressed within the cytosol and nuclei of cells from a variety of tissues. It serves as a tumor suppressor and transcriptional repressor that is highly conserved from Drosophila to mammalian species. VHL binds to Elongin B and C subunits and other proteins as part of a E3 ubiquitin ligase complex that targets a variety of proteins, including HIF-1 alpha and Beta-2 adrenergic receptors, for proteasomal degradation. Aberrant VHL expression is associated with a number of diseases including Von Hippel-Lindau disease, a familial cancer syndrome, that predisposes individuals to hemangioblastomas of the central nervous system and retina, pheochromocytoma and renal cell carcinoma. Three distinct VHL isoforms ranging in molecular weight from approximately 19-30 kDa have been described. Western blot analysis of VHL expression and RNAi validation of anti-VHL antibody specificity - Panel 1. Lysate prepared from HeLa S3 cells (Panel 1a) was blotted using Purified Mouse Anti-VHL antibody (Cat. No. 564183) at concentrations of 1 (lane 1), 0.5 (lane 2), or 0.25 (lane 3) μg/mL. VHL was identified as a band of ~24 kDa (Panel 1a). Lysates from untreated, VHL RNAi- or ERK2 RNAi-treated HeLa S3 cells (Panel 1b) were blotted with Purified Mouse Anti-VHL or Anti-ERK-2 (Cat. No. 554095) antibody. The expressed level of VHL protein blotted with Anti-VHL antibody (2 μg/mL) is downregulated in lysate prepared from HeLa S3 cells treated with VHL RNAi but not ERK2 RNAi. Similarly, ERK2 protein expression was reduced in lysate from HeLa S3 cells treated with ERK2 RNAi but not VHL RNAi when blotted with Anti-ERK2 antibody (0.5 μg/mL). Immunohistochemical staining of VHL expressed in tissue from a human renal tumor - Panel 2. Following antigen retrieval with BD Retrievagen A buffer (Cat. no. 550524), the formalin-fixed paraffin-embedded sections were stained with either Purified Mouse IgG2b, κ Isotype Control (Cat. No. 557351, Top Tissue) or Purified Mouse Anti-VHL antibody (Bottom Tissue) using the BD Biosciences Protocol for Immunohistochemical Staining. A three-step staining procedure that employs a Biotin Goat Anti-Mouse Immunoglobulin (Cat. No. 550337), Streptavidin-Horseradish Peroxidase (HRP) (Cat. No.550946), and the DAB Substrate Kit (Cat. No. 550880) was used to develop the primary staining reagents. The tissue was counterstained with Hematoxylin. Original magnification: 20X. Flow cytometric analysis of VHL expression in VHL-transfected 293F cells - Panel 3. Untransfected (dashed line histogram) and human VHL-transfected (solid line histogram) 293F cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) and permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050). The cells were then washed and stained with Purified Mouse Anti-Human VHL antibody followed by APC Goat Anti-Mouse Ig (Cat. No. 550826) using BD Biosciences Protocol for Intracellular Staining. The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact cells. Flow cytometric analysis was performed using a BD FACSCanto™ II Flow Cytometer System. 564183 Rev. 1 Page 1 of 2
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Antigène
VHL
(Von Hippel-Lindau Tumor Suppressor, E3 Ubiquitin Protein Ligase (VHL))