Mouse IgG2a Isotype Control (M2A)

Western Blot: Mouse IgG2a Isotype Control (M2A) [NBP1-96778] -

FASN and CAD Binding Specificity of pVHL Mutants Derived from VHL Patients. (A) Schematic representation of pVHL domains. Dotted lines indicate the approximate positions of the HIF-1 alpha and Elongin C binding domains. The alpha and beta structural domains are also indicated. (B) Effects of pVHL mutations on interactions with E3 ligase components, HIF-alpha and other interacting proteins including FASN. pVHL mutants were stably expressed by lentiviral vectors in HCT116 cells, and WCEs were tested in precipitation assays. Note that the mutant pVHL-W88S almost completely lost the ability to bind to HIF-1 alpha and HIF-2 alpha, whereas pVHL-Y112H retains this ability almost completely. However, both pVHL-W88S and pVHL-Y112H show significantly reduced binding to FASN and CAD, as indicated by small and large asterisks, respectively. Arrows indicate HIF-1 alpha and HIF-2 alpha bands. Long arrows and arrowheads indicate unmodified and modified forms of FASN, respectively. Protein samples prepared from hypoxia (1% oxygen)-exposed cells were added as positive control in left-end extra lanes. (C) The same samples as used in B were analyzed by silver staining. The E3 ligase components appear as major bands as indicated. (D) Subcellular localization of stably expressed pVHL mutants in 786-O. pVHL-Y112H and pVHL-W88S showed preferential nuclear localization, whereas pVHL-WT and pVHL-H115Q which both strongly bind to FASN and CAD, showed cytoplasmic localization. Cells showing clear nuclear (C  N) or cytoplasmic (C > N) localization in three microscopic viewing areas were counted as shown in the lower panel. The total counted cell numbers ranged from 35 to 64. The experiments were repeated more than twice, except for the cell localization count, and the results were reproduced. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28775317), licensed under a CC-BY license. Not internally tested by Novus Biologicals.