E, F, G, H Pulse-chase diUb synthesis assays using different E2s, UBE2S (E), UBE2C (F), UBE2D2 (G), or UBE2R2 (H). DiUb formation was monitored by fluorescent scanning (top), quantitated (bottom), and assessed for statistical significance by either an unpaired Welch's t-test (UBE2C and UBE2R2) or one-way ANOVA (UBE2S and UBE2D2) (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, n=3 independent, technical replicates for all E2s). Error bars: SEM. Data normalized to the maximal WT diUb formation. # denotes a contaminant band. 