Inhibition of Triad1 expression results in increased Gfi1 ubiquitination. (A) HEK293 cells were not transfected or transfected with indicated siRNAs against Triad1 or a control siRNA (scrambled control; Qiagen) and GFP. At 48 hours after transfection, GFP⁺ cells were sorted by FACS and equal amounts of cells were lysed and stained for Triad1 expression, showing efficient down-modulation of endogenous Triad1. The same blots were probed with an actin antibody to demonstrate equal loading. (B) In vivo ubiquitination experiments in HEK293 cells showed that Triad1 siRNA expression resulted in increased polyubiquitination of Gfi1. Cotransfection with a control siRNA (left panel, siRNA against Sp1; right panel, scrambled siRNA; Qiagen) had no effect on Gfi1 ubiquitination. wce indicates whole-cell extract.