FIG. 8.
Solution oligomerization of GR in mammalian cells is independent of the targeting of GR to DNA. (A) The new constructs (GFP-GRR496H, buGR505C, and myGR505CNl1−) used in this experiment are summarized schematically. A schema of the myGRNL1− construct is shown in Fig 7. (B) In situ immunofluorescence analysis of the localization of GFP-GRR496H, myGRNL1−, buGR505C, and myGR505CNL1− expressed singly and in the combinations shown. Dex treatment at 10−6 M for 1 h was performed as indicated. Specific localization of the GR constructs was visualized as follows: GFP-GRR496H using direct fluorescence; myGRNL1− by indirect immunofluorescence analysis using antibody 9E10 followed by a rhodamine-red-conjugated anti-mouse secondary antibody (allowing the detection of myGRNL1− in the presence of GFP-GRR496H); buGR505C by indirect immunofluorescence using antibody BuGR2 and a fluorescein-conju- gated anti-mouse secondary antibody; myGR505CNL1− by indirect immunofluorescence using antibody 9E10 followed by a fluorescein-conjugated anti-mouse secondary antibody. Photomicrographs of the immunofluorescence pattern of representative cells are shown to the left, while quantification of observations of a minimum of 150 cells for each sample in each of a minimum of three independent experiments performed in triplicate is shown to the right. The localization of myGRNL1− prior to steroid treatment is shown in Fig. 7 and is not repeated here. GR localization in each cell was categorized as completely or mostly nuclear (solid grey bars), equally distributed throughout the cell (stippled bars), or localized predominantly or exclusively to the cytoplasm (white bars). The error bars indicate the standard errors of the means. Bar, 10 μm.