Figure 3.
TMD-Dependent mCD4 Ubiquitination with Purified Factors
(A) Diagram depicting the purified system.
(B) Coomassie-stained gels of recombinant purified proteins.
(C) Co-IP analysis of recombinant Vpu and mCD4 in reconstituted proteoliposomes. Radiolabeled mCD4 and recombinant Vpu were incorporated either separately or together into liposomes, and the indicated samples were mixed, solubilized, and subjected to IP with anti-Vpu or control IgG. The samples before and after IP were analyzed by immunoblotting for Vpu and autoradiography for mCD4, respectively.
(D) Liposomes reconstituted with mCD4 alone, with Vpu (WT), or with Vpu-SN were incubated with CK2 (as indicated) and ubiquitination factors (E1, UbcH3, UbcH5c, Myc-ubiquitin, ATP). mCD4 was immunoprecipitated and the samples blotted for ubiquitin or mCD4.
(E and F) Vpu and mCD4 combinations as depicted in (E) were subjected to ubiquitination and analyzed as in (D).