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

Figure 8

pCAF enhances K48, K63, and monoubiquitination of CIITA. (a) In vivo ubiquitination assay. COS cells were cotransfected with Myc-CIITA, Flag-pCAF, HA-K48 ubiquitin, HA-K63 ubiquitin, or HA-mono Ub. Eighteen hours following transfections, MG132 was added to indicated samples to inhibit the 26S proteasome in treated cells. Cells were harvested, lysed, precleared, and immunoprecipitated with anti-Myc antibody. Western blots were performed and immunoprecipitated samples were immunoblotted using anti-HA antibody. Lysate controls (bottom two panels) demonstrate expression of Myc-CIITA and Flag-pCAF. Data shown are cropped images from one immunoprecipitation gel and one lysate gel and are representative of three individual experiments.

(a)

(b)

(c)