Triad1 is a Ub ligase. (A) Human Triad1 interacts with UbcH7 in yeast 2-hybrid assays as shown by growth on double histidine and adenine selection (left). The Triad1 mutants H158A and C161A fail to bind to UbcH7 (right). Used bait (left) and prey-constructs (right) are indicated. EV indicates empty vector control. (B) GST-Triad1 efficiently captures UbcH7 from human NB4 cell lysates, whereas GST alone does not. (C) Cotransfection of Triad1-myc and Flag-Ub followed by myc immunoprecipitation (IP) and Flag staining detects a smear of ubiquitinated proteins [X-(ub)_n] bound to Triad1. The amount of ubiquitinated proteins bound to Triad1 was more intense when cells were treated with a proteasome inhibitor (12.5 μM MG132). (D) Cotransfection of GFP-Triad1 and Flag-Ub followed by GFP IP. Immunoprecipitates were split in 2 parts and run on the same gel as indicated and independently stained for Triad1 and Flag, respectively, revealing GFP-Triad1 (arrowhead) and a smear of ubiquitinated proteins ([X-(ub)_n], including proteins with sizes smaller compared with Triad1. Asterisk represents immunoglobulin H (IgH). (E) In vitro Ub ligase assay. Reticulocyte fraction IIA, Triad1 (0.03 μg) or 10^*Triad1 (0.3 μg) without Ub (-), with wild-type Ub (WT) or UbK48-only (K48) were incubated followed by staining of ubiquitinated proteins. Note the marked increase of both wild-type and K48-only ubiquitinated species after addition of Triad1. Increased dose of Triad1 correlates with increase in ubiquitinated proteins. Equal protein transfer between the tested conditions is indicated by the Ub band (bottom).