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Fig. 6. 14-3-3 binding to the tetrameric CD4pLI reporter proteins correlates with their cell-surface expression. Co-immunoprecipitation of 14-3-3 proteins and indicated CD4pLI-fusion proteins. HEK293T cells transiently transfected with the specified constructs were treated with the crosslinker DSS where indicated. (A) Cell extracts (2% of input) treated with DSS are shown to demonstrate the degree of crosslinking as evident from the ratio between 14-3-3 monomer (lower band) and dimer (upper band). Note that the anti-14-3-3 ß antibody recognizes all 14-3-3 isoforms. M indicates molecular mass in kDa. (B) Anti-CD4 immunoprecipitates (upper panel, 90%, lower panel 10%) were resolved by SDS-PAGE. Western blots were probed with antibodies to 14-3-3 proteins (upper panel) and CD4 (lower panel) to confirm equal precipitation of the membrane proteins in all samples. The position of the weakly cross-reacting IgG heavy chain (IgG hc) is indicated in the upper blot. Note that 14-3-3 co-immunoprecipitation was detected using a maximum-sensitivity substrate for HRP indicating that the 14-3-3-bound population of CD4pLI is small or difficult to solubilize. The relative efficiency of 14-3-3 co-immunoprecipitation with the indicated constructs was, however, identical in three independent experiments. The rabbit anti-14-3-3 ß antibody employed for detection recognizes all 14-3-3 isoforms.
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