Figure 6.

(A) Co-immunoprecipitations in PC3 cells for detecting HIF interactions. Transfection of PC3 cells with receptors ERα, ERα-Trunc, ERβ1, ERβΔCX (Same as ERβ2 but lacks the c-terminal spliced in peptide unique for ERβ2), ERβ2 and ERβ5 fused to biotinylation consensus, together with biotin ligase expressing plasmid BirA and expression plasmid for HIF-1α or HIF-2α. After 24 hours, cell extracts were made and biotinylated proteins were bound to streptavidin magnetic beads for 2 hours. Beads were washed three times for 10 minutes in lysis buffer, then the beads were boiled in SDS loading buffer and proteins were separated on SDS-PAGE. Co-immunoprecipitated HIF-1α protein was detected on western blot and on a separate blot biotinylated receptors were detected using Streptavidin-HRP. (B) The same procedure was repeated as in (A) exchanging HIF-1α for HIF-2α, and detecting with HIF-2α antibody.