UbcH7 conjugated with WT‐Ub, I44A‐Ub, or V70A Ub as indicated was incubated with either H887A‐HOIP
RBR
‐
LDD (left) or H359A‐HHARI
RBR (right) mutants that allow trapping of the E3˜Ub thioester with WT‐Ub
29,
41. While a HOIP˜Ub thioester species is observed when UbcH7 was charged with WT‐Ub, no detectable transfer occurs with the I44A‐Ub conjugate (left). Similarly, V70A‐Ub shows reduced formation of the HHARI˜Ub thioester (right). In addition to blotting for HA‐Ub, the blot on the right was also blotted for GST‐HHARI.