FIG 8.

wt ORF2 but not the ORF2 mutants cooperate with HMGA1 to stimulate β-catenin-dependent transcription. (A) Schematic of ORF2 and positions of NLS and PKA/PKC phosphorylation sites. The ORF2-stop construct lacks the first in-frame methionine codon and contains three stop codons at the beginning of ORF2. The ORF2-ΔNLS construct lacks the nuclear localization signal and localizes to the plasma membrane in transfected Neuro-2A cells. The ORF2-P construct contains alanine substitutions in the 5 consensus PKA or PKC phosphorylation sites. (B) Neuro-2A cells were transfected with the designated plasmids (1.0 μg DNA) using the Lipofectamine 2000 transfection reagent. At 48 h after transfection, the cell lysate was prepared and the levels of ORF2 expressed by the respective plasmids were determined by Western blotting using the Flag antibody. (C) Neuro-2A cells were transfected with the designated plasmids using Lipofectamine 2000. The efficiency of transfection of Neuro-2A cells with Lipofectamine 2000 was less than that with the TransIT Neural reagent used in the assay whose results are presented in Fig. 7; consequently, the activation levels were also lower. However, the overall trends were the same. The amount of plasmid DNA in each transfection was the same as that in the empty expression plasmid (pCMV2-Tag-2B) used to construct the ORF2-expressing plasmid. Dual-luciferase assays were performed at 48 h after transfection. The results for cells transfected with 0.1 μg of the Top-Flash luciferase reporter and the Renilla luciferase under the control of a minimal herpesvirus TK promoter (0.05 μg DNA) were normalized to a value of 1, and the fold activation was calculated for the other samples. These results are the averages from three independent experiments. *, a significant difference (P < 0.05) compared to the result for the indicated control by one-way analysis of variance and Fisher's least-significant-difference multiple-means comparison tests.