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Fig. 2. PMA-dependent regulation of GFP-ß2-chimaerin in intact Jurkat cells. (A) Jurkat T cells were transfected with vectors encoding GFP-ß2-chimaerin C1 domain (GFP-C1), GFP-ß2-chimaerin, GFP-ß2-chimaerin Q32A and GFP-ß2-chimaerin I130A. At 24 hours post-transfection, cells were suspended in HBSS and plated on poly-D,L-lysine-coated chamber slides. Slides were mounted on a 37°C plate and analyzed by confocal microscopy. PMA was added at 100 ng/ml after the first frame, and images captured every 10 seconds. Time-lapse confocal recording is shown in Movies 1-3 in the supplementary material. Images are shown at the indicated times to illustrate subcellular protein localization. (B) Jurkat T cells were transfected with GFP-ß2-chimaerin or GFP-ß2-chimaerin C1 domain mutant (GFP-F215G). At 24 hours post-transfection, cells were treated as in A. PMA (800 ng/ml) was added after the first frame, and images captured every 10 seconds. Time-lapse confocal recording is shown in Movie 4 in supplementary material. Images are shown at the indicated times to illustrate subcellular protein localization. (C) Jurkat T cells were co-transfected with a EE-tagged, constitutive active (RacV12) Rac mutant and either GFP-ß2-chimaerin or GFP-F215G mutant. At 24 hours post-transfection, cells were stimulated with PMA (100 ng/ml) for 3 minutes, fixed, stained with anti-EE antibodies (red) and images acquired by confocal microscopy. GFP-ß2-chimaerin localization (green) is shown in the left column, RacV12 expression (red) is shown in the middle column, merge images are shown on the right. Time-lapse confocal recording of a similar experiment performed in living cells is shown. Bars, 5 µm.
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