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Figure 9


Fig. 9. Subconfluent MDCK cells expressing Fc38-63 were transfected with the caveolin 1 siRNA-expressing vector. Forty-eight hours post-transfection, the cells were incubated with anti-Fc receptor antibodies, Ctx-B conjugated to Alexa Fluor-594 or transferrin conjugated to Alexa Fluor-594 for 30 minutes at 4°C. The cells were then washed and shifted to 37°C for 2 minutes. At this time, the cells were shifted to citrate buffer, pH 1.5, for 5 minutes at 4°C to elute surface-bound molecules. The cells were then rinsed in PBS and fixed. Following permeabilization, cells that had been incubated with anti-Fc receptor antibodies were incubated with anti-rat IgG conjugated to Alexa Fluor-594. Immunofluorescent molecules were visualized on a Zeiss LSM 510 confocal microscope. The bar graph in B has quantified the effect of the caveolin 1 siRNA on the endocytosis of Fc38-63 and transferrin in 20 randomly chosen cells. The effect of a control siRNA directed against EGFP on Fc38-63 endocytosis is also illustrated (B). The standard error in B is indicated. Bars, 10 µm.





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