5884 Nucleic Acids Research, 2011, Vol. 39, No. 14 indicating thereby speciﬁc recruitment of HSF1 and Nef at this site. These results further potentiate our ﬁnding that HSF1 is necessary for Nef induced HSP40 gene expression. Nef interacts with human HSF1 both in vitro and in vivo In order to test whether the co-recruitment of Nef and HSF1 on HSP40 promoter in vivo was a result of their physical interaction, we performed co-immunoprecipita- tion with lysates of 293T cells expressing Flag-HSF1 and HA-Nef. The Nef protein co-immunoprecipitated with human HSF1 (Figure 3A, lane 3) in cells expressing both the proteins. Similar co-immunoprecipitation experi- ment with HIV-1-infected CEM-GFP cell lysates also showed interaction of HSF1 with Nef in infected cells (Figure 3B). We have also looked at the possible interaction of Nef with Sp1 in HIV-1-infected CEM- GFP nuclear extract, but we did no ﬁnd any interaction between Nef and Sp1 (Figure 3C). Thus both HSF1 and Nef seem to interact in HIV-1-infected cells and might exist as a complex on HSP40 promoter for its induction. To further identify the HSF1 interacting domain of Nef, puriﬁed His-HSF1 was incubated with 293T cell lysates expressing Nef wild-type or mutant proteins followed by pull down of complex by immobilization on Ni-NTA beads (Figure 3D). As expected Nef protein was speciﬁc- ally pulled down by His-HSF1. Although there was some reduction in binding with all the nef mutants but mutation in proline-rich motif of Nef between amino acids 69–78 completely abolished its interaction with HSF1 (lane 5, Figure 3D). This result further conﬁrms that Nef and HSF1 speciﬁcally interact with each other and proline Figure 3. HIV-1 Nef physically interacts and co-localize with HSF1 both in vitro and in vivo. (A) HSF1 and Nef interact in 293T cells overexpressing HA-Nef and Flag-HSF1 as analyzed by co-immunoprecipitation. The input indicates lysate prepared from 293T cells co-transfected with HA-Nef and Flag-HSF1 expression vectors. (B) Nef co-immunoprecipitates with HSF1 in HIV-1-infected CEM-GFP cells. The input indicates lysate prepared from CEM-GFP cells infected with HIV-1. (C) Nef and Sp1 do not interact in HIV-1-infected CEM-GFP cells. The input indicates lysate prepared from CEM-GFP cells infected with HIV-1. (D) Proline-rich motif of Nef is important for interaction with HSF1. Lysates of HEK-293T cells expressing different HA-tagged Nef and its mutants were used for His-pull down with puriﬁed His-tagged HSF1. The input indicates the lysates prepared from wild-type and mutant Nef transfected 293T cells. (E) Nef and HSF1 co-localize in Nef and HSF1 expressing cells. Immunoﬂuorescence studies were performed with 293T cells transfected with Nef and HSF1 vectors (left column), pNL4-3 transfected 293T cells (middle column) and HIV-1-infected Jurkat cells using Nef and HSF1 antibody (right column). The bottom panel in each column is a magniﬁed image of one cell from the merged image panel. Arrows indicate co-localization of Nef and HSF1.