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Fig. 3. Subcellular fractionation of COS7 cells expressing the various GFP constructs. (A) Transfected COS7 were lysed and after clarification of the cellular debris by centrifugation, fractionated into supernatant (S) and pellet (P) fractions by ultracentrifugation for 16 hours at 200,000 g as described in the Materials and Methods section. The fractions were subjected to a SDS-PAGE, analysed by western blot with an antibody against GFP and the resulting bands were quantified using UVIband V97 software. In addition, COS7 cells transfected with the tagged GFP constructs were extracted in the presence of Triton X-100 at 4°C and subjected to centrifugation on a 40:30:5% sucrose gradient. After centrifugation, the gradient tubes were divided into 12 equal aliquots collected from the bottom and analysed by SDS-PAGE and western blot (lower panels). (B) COS7 cells were transfected with the Myr, Myr (1-94) and A2C (1-94) NOS2-GFP. The bottom right panels are the merges of the three fluorescence signals. Bars, 50 µm.
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