FIG. 3.

Enhanced coactivation of the LMP1 promoter by DP103 binding-deficient SMN ΔEx6/7. (A) Mapping of the DP103 binding site on SMN. 293GP cells were transfected with pSG5 constructs encoding HA-tagged SMN mutants (10 μg) as indicated. After 36 h native cell extracts were immunoprecipitated with anti-HA MAb 3F10 (IP: anti HA and control 2) or unspecific MAb 3A6 (controls 1 and 3) and analyzed by SDS–10% PAGE and Western blotting. Precipitated transfected SMN mutants were detected by using anti-HA MAb 3F10 (WB: anti HA), and coprecipitated endogenous DP103 was detected by using anti-DP103 MAb 8H4 (WB: anti DP103). The positions of the molecular mass markers (in kilodaltons) are indicated on the left side of each panel. Deletion of SMN exon 6 abolished coprecipitation of endogenous DP103. (B) Schematic representation of the SMN mutants tested. (C) Coexpression of EBNA2 and the HA-tagged DP103 binding-deficient SMN mutant SMN ΔEx6/7 further increased coactivation of the −327/+40 LMP1 promoter luciferase construct. Assays were performed as described for Fig. 1B. Graphs represent the mean values of three independent experiments performed in duplicate (±SEM). (D) Immunofluorescence of HA-tagged WT SMN (a) and SMN ΔEx6/7 (c) expressed in HeLa cells and stained with 3F10 anti-HA/anti-rat TRITC MAbs. (b and d) Nuclei were visualized by using DAPI (4′,6′-diamidino-2-phenylindole). Loss of binding to DP103 released SMN ΔEx6/7 from nuclear gems/coiled bodies. (E) Enhanced colocalization of EBNA2 and DP103 binding-deficient SMN ΔEx6/7. HeLa cells coexpressing EGFP-EBNA2 (a) and HA-tagged SMN ΔEx6/7 (b) were stained by using anti-HA 3F10/anti-rat TRITC MAbs and subjected to confocal laser scanning microscopy. In the merged image (subpanel c), colocalization results in a yellow signal.