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. 2009 Sep 4;106(38):16275–16280. doi: 10.1073/pnas.0904305106

Fig. 4.

Fig. 4.

p300/CBP E3 activities are predominantly cytoplasmic. (A) (Top and Middle) Nuclear and cytoplasmic fractions of U2OS cells were immunoprecipitated with anti-CBP antibody, and the washed IPs incubated with E1/E2, Ub, and ATP as indicated, followed by anti-Ub and CBP immunoblotting of the reactions. (Bottom) Immunoblot of CBP and Rb (nuclear marker) in nuclear and cytoplasmic fractions. (B) E4 activity of cytoplasmic CBP. p53/MDM2 complexes (insect-cell-derived) were incubated with E1/E2, Ub, or methyl-Ub, ATP and CBP IPs from control-sh or CBP-shA cytoplasmic fractions as indicated, followed by anti-p53 (Top) or anti-CBP (Middle) immunoblotting of the reactions. Migration positions for native, monoubiquitinated/MUM and polyubiquitinated p53 species are indicated. (Bottom) Relative abundances of native, mono-Ub/MUM, and polyubiquitinated p53 species were quantitated by densitometry.