Fig 1: AF facilitates ubiquitin charging to E2s.The effects of AF on ubiquitin charging in vitro to UBA1 (a), UBE2G2 (b, c), and UBE2D1 (d). The assays were performed using purified recombinant proteins. 250 nM UBA1 alone or in combination with 4 µM His-E2s (His-UBE2G2 or His-UBE2D1) were treated with increasing concentrations of AF in reaction buffer for 1 min. ATP (50 µM) was added to initiate the reaction. The reactions proceeded at 15 oC for 45 s and stopped by adding non-reducing loading buffer. 5 mM DTT was used to disrupt the thioester bond that links ubiquitin to the active cysteine of E2s. Source data are provided as a Source Data file.