Fig. 4.

Affinity of HIF-2α mutants for pVHL is correlated with disease phenotype. a Biotinylated HIF-2α peptides (523–541) were immobilized on a streptavidin-coated 96-well plate. Binding to various concentrations of pVHL-elongin B-elongin C (VBC) complex was assayed via ELISA with an anti-eloB antibody. Absorbance was read at 450 nm. Values represent mean of three independent experiments performed in duplicate ± SEM. *p < 0.05; **p < 0.01. b Biotinylated HIF-2αOH peptides (523–541) were immobilized on streptavidin- agarose beads and incubated with in vitro transcribed and translated (IVTT) pVHL. Streptavidin beads were pulled down (PD) and levels of HA-tagged pVHL were visualized via immmunoblotting (IB). c, d Biolayer interferometry kinetic analysis of VBC complex binding to biotinylated HIF-2α peptides (523–541). c Biotinylated peptides were coupled to streptavidin- coated biosensors and monitored for binding to VBC complex at the indicated concentrations. The data were analyzed based on a 1:1 binding model using the BLItz Pro software with the fitted curves shown as gray lines. Sensorgrams are representative of three experiments conducted with independently purified protein. d Rate plane with Isoaffinity Diagonals (RaPID) plot highlighting the kinetic parameters of VBC complex binding to HIF-2α peptides. Values represent mean of three experiments conducted with independently purified protein ± SEM. * denotes uncharacterized band