Figure 7.

Monoubiquitinated UBCH5 forms a stable complex with SMURF2. (A and B) Purified GST-SMURF2 and recombinant UBCH5 (in A) and purified GST-SMURF2, UBCH5, and the ubiquitination machinery including E1, ubiquitin, and ATP (in B) were mixed, incubated, and subjected to analytical SEC. The top panel is a representative spectrum. In the bottom panel, indicated fractions were subjected to immunoblot analysis using indicated antibodies. The presence of both SM2 and UBCH5 in the same fraction indicates the formation of a stable complex. (C) HEK293 cells were transfected with Myc-tagged UBCH5 in the presence or absence of FLAG-tagged SM2 wild type. Twenty-four hours post-transfection, cells were treated with 3-MA in the indicated samples, and 4 hours post-treatment, lysates were prepared. Cell lysates were then subjected to immunoprecipitation using Myc-agarose beads and immunoblotted with the indicated antibodies. (D) Purified GST-SM2 (either wild type or CA) and purified UBCH5 was subjected to GST pull-down assay as described in the Materials and Methods section. Immunoblot analysis was performed using indicated antibodies.