FIG 5.

Examination of ORF2 with β-catenin or HMGA1 in transiently cotransfected Neuro-2A cells. Neuro-2A cells were cotransfected with 0.5 μg of two of the following plasmids that express Flag-tagged β-catenin (S33Y) by use of the Lipofectamine 2000 transfection reagent (Invitrogen): a β-catenin construct that was not Flag tagged, HMGA1, or Flag-tagged ORF2. At 48 h posttransfection, cells were fixed and then stained with the designated primary antibodies. (A) The S33Y construct that expresses Flag-tagged β-catenin was stained with the Flag monoclonal antibody (red) and HMGA1 (green). (B) A β-catenin-specific antibody (catalog number ab32572; Abcam) was used to detect non-Flag-tagged β-catenin (green) and Flag to detect ORF2 (red). (C) HMGA1 (green) and Flag to detect ORF2 (red). Nuclear DNA was stained with DAPI. The images were observed by confocal microscopy. The selected images are representative of those from four experiments (at least 100 stained cells were examined). Bars, 10 μm.