|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 5. A mutation in coilin that disrupts PIASy interaction reduces the frequency of CB/PML body association. (A) Coilin knockout MEF cells were co-transfected with myc-PIASy and various GFP-tagged control or mouse coilin constructs. After immunoprecipitation with anti-GFP antibodies and SDS-PAGE, a western blot was conducted with antibodies to the myc-tag (upper panels). The blot was reprobed with anti-GFP to verify that GFP, GFP-mouse coilin (coilin), GFP-mouse coilin (
100-150) and GFP-mouse coilin (150-200) were immunoprecipitated (middle and lower panel). The position of the immunoglobulin heavy chain, IgG(H), is noted. (B) Coilin knockout MEF cells were transfected with full-length and mutant GFP-tagged mouse coilin constructs (green) followed by detection of PML bodies using anti-PML (red). DAPI staining (blue) was used to define the nucleus. Cells were scored for associations between CBs and PML bodies. In panels A-C, the percent in the bottom right of the image accounts for the majority of scored cells. For example, GFP-mouse coilin (panel A) forms CBs that associate with PML bodies (double arrowheads) in 80% of cells. By contrast, 92% of cells with CBs (arrows) formed by GFP-mouse coilin (100-150) (panel B) do not show association with PML bodies (arrowheads). In panels D-G, coilin knockout MEF cells were transfected with GFP-mouse coilin (100-150) + NLS and stained with anti-PML. The percent in the bottom right of each image accounts for the number of cells expressing this protein with similar localization patterns. For example, 49% of cells expressing GFP-mouse coilin (100-150) + NLS (panel D) have cytoplasmic accumulations and CBs (arrows) which do not associate with PML bodies (arrowheads). By contrast, only 11% of transfected cells display a strictly nuclear coilin signal and CBs that associate with PML bodies (panel F, double arrowhead). Scale bar, 2 µm.
|
