Fig. 2: DCAF1 mediates K48-linked polyubiquitination of Rheb.

A Partial result of mass spectrometry analysis of the Rheb-associated immunoprecipitated complex. B, C Co-IP of Flag-Rheb and endogenous DCAF or Flag-DCAF1 and endogenous Rheb. 293 T cells transfected with an empty vector or Flag-Rheb (B) or Flag-DCAF1 (C), followed by IP analysis with anti-Flag and IB analysis as indicated. D, E IP analysis of endogenous DCAF1 and endogenous Rheb. IB analysis of co-immunoprecipitated endogenous DCAF1 and Rheb proteins using anti-Rheb (D) or anti-DCAF1 (E) antibody in Huh7, HCT116, and EC109 cells. An unrelated IgG was used as negative control. F Co-localization analysis of DCAF1 and Rheb. Huh7, HCT116, and EC109 cells were fixed and immunostained with antibodies against the indicated proteins. Representative images are shown. Scale bars: 25 μm. G DCAF1 promotes Rheb ubiquitination. 293T cells were transfected with recombinant plasmids as indicated and treated with 10 µM MG132, and subjected to IP with anti-Flag, followed by IB with anti-HA or anti-Flag. H, I Knockdown of DCAF1 attenuates Rheb polyubiquitination. H 293T cells were transfected with siDCAF1, Flag-Rheb, and HA-Ub as indicated, and subjected to IP with anti-Flag, followed by IB with anti-HA. I Huh7 cells were transfected with siDCAF1, and Rheb polyubiquitination was analyzed by IP with anti-Rheb, followed by IB with anti-Ub. J DCAF1 elevates the K48-linked, but not the K63-linked, polyubiquitination of Rheb. 293T cells were transfected with recombinant plasmids as indicated, treated with MG132, and subjected to IP with anti-Flag, followed by IB with anti-K48 Ub or anti- K63 Ub. All data were representative of at least three independent experiments (n = 3).