Pulling a Ligase out of a “HAT”: pCAF Mediates Ubiquitination of the Class II Transactivator

Figure 3

CIITA ubiquitination depends on the E3 ligase domain of pCAF. (a) The E3 ligase null mutant pCAF negates CIITA ubiquitination. In vivo ubiquitination assay. COS cells were cotransfected with Myc-CIITA, Flag-pCAF, and Flag-pCAF Δ E3 ligase mutants as indicated. Eighteen hours following transfections, cells were harvested, lysed, precleared, and immunoprecipitated with the anti-Myc antibody. Western blots were performed and immunoprecipitated samples were immunoblotted using antiubiquitin antibodies. Lysate controls (bottom two panels) demonstrate expression of Myc-CIITA, Flag-pCAF, and Flag-pCAF Δ E3. Data shown are cropped images from one immunoprecipitation gel and one lysate gel and are representative of three independent experiments. (b) Densitometry and quantification of data in Figure 3(a). Densitometry was performed on three independent experiments, mean ± SD, . Area for densitometry analysis was selected from CIITA’s molecular weight of 150 KDa as labeled and above.

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