FIG. 2.

(A) Dose-dependent coactivation of the −327/+40 LMP1 promoter by coexpression of EBNA2 and increasing amounts of HA-tagged SMN (indicated in micrograms). Assays were performed as described for Fig. 1B. Graphs represent the mean values of three independent experiments performed in duplicate (±SEM). (B) SMN increases EBNA2-mediated induction of endogenous LMP1 protein. EBV-positive P3HR1 cells were transfected with pSG5 constructs (15 μg) encoding EBNA2 or HA-tagged SMN, as indicated. Cells were harvested and subjected to SDS–10% PAGE and Western blotting by using MAbs S12 (anti-LMP1), R3 (anti-EBNA2), 3F10 (anti-HA), and anti-β-actin MAb (Sigma). The positions of the respective proteins are indicated by arrows. The positions of the molecular mass markers (in kilodaltons) are indicated on the left side. (C) Subcellular distribution of EBNA2, DP103, and SMN. HeLa cells transfected with pSG5-HA (red signals) or pEGFP-C1 (green signals) constructs (5 μg) encoding the corresponding fusion proteins of EBNA2, DP103, or SMN were immunostained and analyzed by confocal laser scanning microscopy. HA-tagged proteins were visualized by using anti-HA 3F10/anti-rat TRITC MAbs. The localizations of coexpressed SMN and DP103 (upper panel), EBNA2 and SMN (middle panel), or EBNA2 and DP103 (lower panel) are shown. In the merged images, colocalization results in a yellow signal. (D) Subcellular localization of endogenous SMN and EGFP-EBNA2 in BJAB cells. BJAB cells mock transfected (a) or transfected with 10 μg of pEGFP-C1 EBNA2 (b, c, and d) were immunostained by using anti-SMN 7B10/anti-mouse TRITC MAbs and subjected to confocal laser scanning microscopy. In the merged image (subpanel d), colocalization results in a yellow signal.