FIGURE 5:

DTX3L regulates ESCRT-0 ubiquitination and complex integrity. (A, B) DTX3L regulates the ubiquitination status of ESCRT-0 subunits HRS and STAM-1. HeLa cells were transfected with siRNA directed against control or DTX3L plus FLAG-ubiquitin and T7-HRS (A) or T7-STAM-1 (B). Denatured cleared lysates (300 μg) were incubated with an anti-T7 polyclonal antibody for 1 h and then protein G agarose for 1 h. Bound proteins were eluted in 2× sample buffer and resolved by 7% SDS–PAGE and analyzed by immunoblotting with the indicated antibodies. Immunoblots are representative of three independent experiments. Data were quantified by densitometric analyses and followed by Student's t test. Ubiquitination of T7-HRS or T7-STAM-1 in control siRNA–treated samples was normalized to 1 and compared with ubiquitination in the DTX3L siRNA–treated samples. Error bars represent SEM. (C, D) DTX3L regulates the integrity of ESCRT-0. HeLa cells were transfected and siRNA directed against control or DTX3L. Cleared cell lysates (300 μg) were incubated with control IgG and HRS (C) or STAM-1 (D) antibodies. Bound proteins were eluted in 2× sample buffer, resolved by 7% SDS–PAGE, and analyzed by immunoblotting with the indicated antibodies. Immunoblots are representative of three or four independent experiments. Data were quantified by densitometric analysis, and control siRNA–treated samples were normalized to 1 for either HRS or STAM-1 and compared with binding in the DTX3L siRNA–treated samples. Data were analyzed by Student's t test.