Fig. 4: RSUME potentiates VHL mutants loss of function by decreasing VHL–HIF interaction and ECV complex assembly.
From: von Hippel-Lindau mutants in renal cell carcinoma are regulated by increased expression of RSUME
a–d COS-7 cells were transfected with 0.5 µg of Flag-VHL variants, 0.5 µg of V5-RSUME, 0.5 µg of HA-HIF-2α, 0.1 μg of Gam-1, Gam-1MUT control vector. a Forty-eight hours post transfection cells were incubated in 2% serum, 5 μM MG-132 for 6 h and lysed. A first immunoprecipitation (IP) using anti-FLAG antibody was performed. Immunoprecipitated fractions were eluted with soluble FLAG peptide and a second IP using anti-V5 antibodies was performed. Immunoprecipitated fractions from the second IP “Tandem IP” and extract aliquots (Input) were analyzed by WB using the indicated antibodies. b–d Cells extracts were immunoprecipitated with anti-FLAG antibodies. Immunoprecipitated fractions and extract aliquots (Input) were analyzed by WB using the indicated antibodies. One representative experiment from three independent experiments with similar results is shown. e Cells were transfected with 0.5 μg of the indicated Flag-VHL-GFP variants, 0.5 μg of Flag-Elongin C, 0.5 μg of HA-Elongin B, 0.5 μg of Cullin-2 and/or 0.5 μg of V5-RSUME expression vectors. Forty-eight hours post transfection cells were lysed and lysates immunoprecipitated with GFP antibody. Immunoprecipitated fractions and extract aliquots (Input) were analyzed by WB using the indicated antibodies. One representative experiment from two independent experiments with similar results is shown
