Truncations of ORTH1 (name and schematic form under their respective western blots) were expressed as GST fusions and tested for in vitro ubiquitylation activity.
(a-e) Top, anti-ubiquitin immunoblot to detect ubiquitylated products; bottom, anti-GST to visualize E3 ligase. Reactions lacking either E1, E2 or E3 (-E1, -E2, -E3, respectively) are compared to comparable reaction with all components (T).
(e) mPHD has the CCHC of the core metal ligands, positions 3-6, all changed to alanine. Full length ORTH1 assays (a) were included for comparison.