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Fig. 10. MDCK cells expressing V5-tagged versions of Fc38-63, Fc38-63Y47A, Fc38-63L50A were lysed and immunoprecipitates were prepared with V5-specific antibodies. The immunoprecipitates were incubated in the absence or presence of a mixture of glycosidases that removes all O-linked and N-linked sugars prior to immunoblotting analysis with V5 antibodies (A). V5 immunoprecipitates prepared from MDCK cells expressing V5-tagged versions of Fc38-63, Fc38-63Y47A, Fc38-63L50A were also subjected to immunoblotting analysis with V5- and caveolin 1-specific antibodies (B). Alternatively, cells expressing V5-tagged (+Tag) and untagged (–Tag) versions of Fc38-63 were incubated with anti-V5 antibodies for 30 minutes at 4°C. The cells were then washed and lysed. Immune complexes were captured with protein A agarose and subjected to immunoblotting analysis with V5- or caveolin 1-specific antibodies (B). V5 immunoprecipitates prepared from MDCK cells expressing V5-tagged AE1-4 or AE1-4L50A were also processed for immunoblotting analysis with AE1- and caveolin 1-specific antibodies (C). Finally, MDCK cells expressing the wild-type (WT) and mutant V5-tagged Fc38-63 or V5-tagged versions of AE1-4 or AE1-4L50A were lysed in 2 ml of isotonic buffer containing 1% LubrolWX. The lysates were fractionated on discontinuous sucrose gradients, and 1 ml fractions were collected from the top of the gradient (fraction 10 includes the pellet). These samples were either directly analyzed by immunoblotting analysis using caveolin 1, furin, or 
-COP antibodies (D), or immunoprecipitates were prepared from each fraction using V5-specific antibodies. The immunoprecipitates were then analyzed by immunoblotting analysis using V5-specific antibodies (D). The top and bottom of the gradient in D are indicated.
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