Co-immunoprecipitation studies with Vpr, DCAF1 and DDB1

Copyright information:Taken from "HIV-1 Vpr activates the Gcheckpoint through manipulation of the ubiquitin proteasome system"http://www.virologyj.com/content/4/1/57Virology Journal 2007;4():57-57.Published online 8 Jun 2007PMCID:PMC1904188. A. Flag-DCAF1 was cotransfected into HeLa cells with either HA-Vpr or HA-Vpr(R80A). Immunoprecipitation was performed with either HA or Flag antibody as indicated. Immunoprecipitates were analyzed by WB for the presence of Flag-DCAF1 or HA-Vpr. B. GFP, HA-Vpr, HA-Vpr(R80A), or HA-Vpr(Q65R) constructs were transfected, and immunoprecipitations with HA antibody were performed. Immunoprecipitates were analyzed by WB with an antibody against endogenous DDB1. C. Knockdown of DCAF1 abolishes the association between Vpr and DDB1; HeLa cells were transected with HA-Vpr, in the presence of non-specific (NS) or DCAF1-specific siRNA. 48 hours later, cell extracts were immunoprecipitated with HA antibody and analyzed by WB for the presence of endogenous DDB1. D. HA-Vpr(Q65R) fails to interact with DCAF1.