Pat1b-GFP localizes to P-bodies while Pat1a-GFP localizes weakly to P-bodies in some cells but exerts a dominant-negative effect on P-bodies in others. (A) Confocal imaging of HeLa cells transfected with hPat1b-GFP or mPat1a-GFP. Cells were fixed 24 h post-transfection and stained with p54 antibodies as a P-body marker. White arrow points to the zoomed P-body (white box). Scale bar, 10 μm. (B) Fluorescent imaging of HeLa cells transfected with mPat1a-GFP showing two phenotypes: cells with P-bodies (a) or lacking P-bodies (b). Cells were fixed 24 h post-transfection and stained with p54 as a P-body marker. (C) Graph of the percentage of cells containing P-bodies (as seen with p54) when transfected with different GFP constructs in HeLa and HEK293 cells. Three independent experiments were performed and standard deviation bars are shown. (NT) Nontransfected cells. (D) The GFP constructs are expressed at similar levels in HeLa cells. Sixty micrograms protein was loaded on a SDS-PAGE gel and analyzed by Western blotting with the indicated antibodies.
