Immunoprecipitation

HEK-293 cells were transiently transfected with expression plasmids encoding Myc-tagged EGFR and V5-tagged PHD2 variants (wt PHD2, catalytically inactive PHD2 (mut H374A), PHD2 Δ1-139aa, and PHD2 Δ208-426aa) to investigate the binding preferences of PHD2 and EGFR. Immunoprecipitations were carried out as described [53]. Cells were harvested 48 h post-transfection, washed twice with ice-cold phosphate buffered saline (PBS) and lysed as described. For immunoprecipitation in the presence of EGF and/or catalytic inhibitors of EGFR and PHD2, cells were pre-treated with AG 1478 (1 mM) or DMOG (2 mM) for 6 h prior to vehicle/EGF (100 ng/ml) treatment for 10 min, then cells were lysed. Aliquots of cleared HEK-293 cell lysates containing 1 mg of total protein were mixed with protein G Sepharose beads (GE Healthcare) and Myc-tagged EGFR was immunoprecipitated with the Myc-Tag antibody (Cell Signaling #2278) or V5-tagged PHD2 proteins were immunoprecipitated with the V5 Tag antibody (Thermo Fisher, R960-25) at 4°C overnight. The next day the beads were washed 5 times with lysis buffer, immune complexes were then resolved on SDS-PAGE 7.5% or 12.5%, respectively and analyzed as below with antibodies against the Myc and V5 epitope. Lysates from EGF and/or inhibitor-treated cells were checked for phosphorylation of EGFR and accumulation of HIF-1α as a verification of the inhibition of PHD2 and EGFR.