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Figure 1


Fig. 1. Ligand-stimulated Eph receptors interact with caveolin-1. (A) Co-fractionation of EphB1 receptor with caveolin-1. Sucrose gradient centrifugations were performed as described previously (Song et al., 1996a). Fractions were collected and subjected to immunoblotting (IB) with antibodies directed against HA-tagged EphB1 receptor (Anti HA) and caveolin-1 (Anti Cav-1). EphB1 receptor and caveolin-1 located to the same, buoyant membrane fractions (#1-3). Immunoblotting with antibodies directed against Src (Anti c-Src) was used to show localization of a cytoplasmic protein, as it was located to the high-density fractions (#6-8). (B) Co-immunoprecipitation (IP) of EphB1 receptor with antibody against caveolin-1 in CHO-EphB1 cells. EphrinB2/Fc was added to the cells for indicated times, then cells were lysed, caveolin-1 (lanes 3-7) and control rabbit IgG (lanes 1-2) immunoprecipitates were resolved by SDS-PAGE and transblotted to PVDF membranes. Membranes were reciprocally immunoblotted with antibodies against HA or caveolin-1, respectively. (C) Immunofluorescence analysis confirmed the association of EphB1 receptor with caveolin-1 on the plasma membrane after 30 minutes of ephrinB2 ligand stimulation (C2) compared with non-stimulated cells (C1). CHO-EphB1 cells were fixed and stained with primary antibodies against HA and caveolin-1 followed by Alexa green- and red-conjugated secondary antibodies, respectively. Samples were processed as described under Materials and Methods. Images were acquired with a Nikon Eclipse E600 microscope, connected to Nikon digital camera DXM1200, and processed with the Nikon AC-1 software version 2.11. Magnification, x1000. (D) Co-immunoprecipitation of EphA2 receptor with antibody against caveolin-1 in PC-3 cells. EphrinA1/Fc was added to the cells for indicated times, then cells were lysed, caveolin-1 (lanes 1-5) and control rabbit IgG (lanes 6-7) immunoprecipitates were resolved by SDS-PAGE and transblotted to PVDF membranes. Membranes were reciprocally immunoblotted with antibodies against EphA2 or caveolin-1, respectively. Results are representative of at least three independent experiments.