Fig. 3. In vitro characterisation of PHD-EGFP fusion proteins. (A) In vitro transcription/translation products detected by 8% SDS-PAGE and autoradiography. (B) An in vitro protein interaction assay. After treatment of an immobilised GalDBD- HIF-1 549-582 fusion protein with unprogrammed reticulocyte lysate or PHD or PHD-EGFP fusion protein, ³⁵S-labelled pVHL was added. Two pVHL isoforms (24 kDa and 19 kDa) were detected because translation was initiated at two distinct ATG codons in VHL. The ability to capture pVHL indicates hydroxylation of HIF-1 Pro564. Note that reticulocyte lysate has a low but significant hydroxylating activity.