Figure 6

TBP and HAP1 co-localization assay in COS-7, 293, and Neuro-2a cells. (A) cells transfected with a single expression plasmid, as indicated at top. Forty-eight hours after transfection, cells were stained with Hoechst 33342 and fluorescent photomicrographs of the blue, red, and green channels were taken. Hoechst stain (blue) shows nuclei. CMVp-GFP-HAP1-A_7–598 was exclusively cytoplasmic in all cells tested, where it assembled into strongly fluorescent STLBs. CMVp-DSRed2-TBP was exclusively nuclear with heterogenous subnuclear distributions when expressed alone. The negative controls, CMVp-DSRed2-PTIP, MED15, and U2AF₆₅ (encoding nuclear proteins unrelated to TBP, see text), were also localized entirely to the nucleus when expressed alone. (B) co-expression of HAP1 with TBP, PTIP, MED15, or U2AF₆₅. Co-expression of CMVp-GFP-HAP1 and CMVp-DSRed2-TBP shows that all HAP1 remained cytoplasmic, where it assembled into STLBs; however, TBP localization was altered. Thus, whereas much of the TBP still localized to the nucleus, a portion was sequestered into GFP-HAP1-STLBs. White arrows designate representative examples of extranuclear TBP in STLBs. As negative controls, DSRed2-PTIP, MED15, and U2AF₆₅ were not detected in GFPHAP1-STLBs.