FIGURE 5.

And-1 prevents the ubiquitination of Gcn5 by CRL4^Cdt2 E3 ligase. A, overexpression of Cdt2 enhances the ubiquitination of Gcn5. U2OS cells transfected with the indicated plasmids were harvested 48 h after transfection. Cells were treated with MG132 for 6 h before harvest. The ubiquitinated species were precipitated by HA antibodies and subjected to immunoblot for ubiquitinated FLAG-Gcn5 using anti-FLAG antibody. Ub, ubiquitin. B, overexpression of And-1 suppresses the ubiquitination of Gcn5. U2OS cells transfected with the indicated plasmids were harvested for 48 h and treated as in A. Note that And-1 overexpression increased the FLAG-Gcn5 protein levels and reduced the ubiquitination of Gcn5. IP, immunoprecipitation; LC, loading control. C, And-1 prevents Gcn5 ubiquitination by CRL4^Cdt2 in vitro. Incubation of immunopurified CRL4^Cdt2 with Gcn5 in an in vitro ubiquitin ligase assay increased the formation of Gcn5 ubiquitinated species. The addition of immunopurified And-1 suppressed the Gcn5 ubiquitination by CRL4^Cdt2. IF, immunofluorescence. D, And-1 overexpression suppresses the ubiquitination of the C terminus of Gcn5. U2OS cells transfected with the indicated plasmids were harvested. FLAG-And-1(1–491) or FLAG-And-1(492–837) was precipitated using anti-FLAG M2-agarose beads. FLAG precipitates and inputs were subjected to immunoblot using anti-ubiquitin, anti-FLAG, or anti-And-1 antibody.