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Fig. 4. Nitric oxide synthesis, subcellular targeting of the NOS2 chimeras at 25°C and in vivo recovery of the subcellular localisation of the wild-type and Myr NOS2 chimeras. (A) COS7 cells were transfected with the wild-type, Myr and C3S NOS2 chimeras and the cells were grown at both 37°C and 25°C. The NOS2 activity was determined at both temperatures using the Griess assay and was represented relative to the wild-type (relative units, RU) at 37°C (usually about 20 µM nitrites). At the same time, the cells grown at 25°C were fixed and analysed by laser confocal microscopy following the GFP fluorescence after excitation at 488 nm. (B) Areas in the plasma membrane with positive fluorescence for GFP (boxed) were selected in the wild-type, Myr (1-94) and A2C(1-94) NOS2 chimeras. The green fluorescence was bleached with the laser and the cell was allowed to recover the plasma membrane fluorescence for 30 minutes. The recovery is representative of three independent experiments. The ratio increment is depicted below. Bar, 50 µm.
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