Interaction of IE1_CORE with PML and TRIM5α.

<p>(A) Enhanced binding of IE1_CORE to PML in coimmunoprecipitation analysis. HEK293T cells were cotransfected with expression plasmids encoding FLAG-hIE1 variants (as indicated) and Myc-PML (isoform VI). Upper two panels: Western blot detection of PML and hIE1 after immunoprecipitation with an anti-FLAG antibody. Lower two panels: detection of PML and hIE1 in cell lysates before precipitation (input). (B) Enhanced binding of IE1_CORE to PML in yeast two-hybrid assays. The graph shows the quantification of β-galactosidase reporter activity after coexpression of PML and full-length hIE1 or the hIE1_CORE(14–382) in yeast cells. (C) The PML coiled-coil is required for IE1 binding in yeast two-hybrid assays. Full-length hIE1 and hIE1_CORE(14–382) were tested for interaction with various C-terminal PML deletion mutants (shown in the upper scheme) by XGal filter lift assays. (D) The PML coiled-coil is sufficient for binding to hIE1_CORE in coimmunoprecipitation analysis. HEK293T cells were transfected with various FLAG-tagged PML deletion mutants, as shown in the upper scheme, together with Myc-hIE1_CORE(1–382). Upper panel: Western blot detection of hIE1_CORE after immunoprecipitation with an anti-FLAG antibody. Lower two panels: Western blot detection of PML variants and hIE1_CORE in cell lysates before precipitation (input). (E) Interaction of hIE1_CORE with TRIM5α. HEK293T cells were cotransfected with expression plasmids encoding Myc-tagged hIE1_CORE(1–382) and either FLAG-tagged PML (isoform VI) or FLAG-tagged rhesus macaque TRIM5α (rhTRIM5α). Upper panel: Western blot detection of hIE1_CORE after coimmunoprecipitation using an anti-FLAG antibody. Lower two panels: Western blot detection of PML, rhTRIM5α and hIE1_CORE in cell lysates before precipitation (input). R, RING domain; B, B-boxes; CC, coiled-coil domain; NLS, nuclear localization signal; S, SUMO.