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

Figure 4

pCAF facilitates CIITA ubiquitination independent of its HAT domain. (a) CIITA is ubiquitinated by pCAF. In vivo ubiquitination assay. COS cells were cotransfected as indicated with Myc-CIITA, Flag-pCAF, and Flag-pCAF Δ HAT mutant. 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 HAT. 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 (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.

(a)

(b)