HIV-1 Rev and HIC interact directly in vitro and form a complex in vivo. (A) GST pull-down assays show that Rev NLS (upper panel) and SV40T NLS (lower panel) interact directly with HIC in vitro . Purified recombinant HIC protein was incubated with immobilised GST-YFP (control) and various GST-YFP fusions proteins (bait). Interacting proteins were subsequently eluted and resolved by SDS-PAGE. HIC was detected by Western Blot analysis and Commassie staining indicated the quantity and quality of GST fusion proteins employed. (B) HIV-1 Rev and HIC form a complex in vivo. 293T cells were transiently transfected with HA-Rev, pFLAG-HIC/-HIC (2-144)/-HIC (144-246). Input and immunoprecipitates were analysed by Western-Blot (WB) to examine expression levels of Rev, HIC and its mutants, and co-immunoprecipitation of HIC, HIC (144-246) and HA-Rev, respectively. Similar to previous studies and for reasons that remain unclear HIC mutant (144-246) expression was difficult to detect in the input [25, 28].