Triad1 is ubiquitinated and targeted for degradation by the proteasome. (A) Cotransfection of Triad1 and His-Ub followed by selection of ubiquitinated proteins using His-select beads under denaturing conditions followed by Triad1 staining detects multiple discrete ubiquitinated Triad1 forms [Triad1-(ub)_n]. Proteasome inhibition (12.5 μM MG132) results in an increase in Triad1 ubiquitination. Left lane contains positive control containing lysate from Triad1 (arrowhead) transduced 293T cells. Asterisk indicates an a-specific band. (B) Cotransfection of Flag-Ub and Triad1 followed by Flag IP and staining for Triad1 detects multiple ubiquitinated Triad1 species (Triad1-(ub)_n) that were more intense when cells were treated with a proteasome inhibitor prior to cell lysis. Note that unmodified Triad1 (arrow) coimmunoprecipitates with ubiquitinated Triad1.