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Figure 2


Fig. 2. EphrinB2 stimulates tyrosine phosphorylation of caveolin-1 at Tyr14. (A) CHO-EphB1 cells were serum starved for at least 24 hours, then stimulated for indicated times with 1-2 µg/ml ephrinB2/Fc. Cell lysates were separated by SDS-PAGE, transferred to PVDF membranes, and blotted with antibodies against phospho-caveolin (p-cav, upper panel) or caveolin-1 (cav-1, lower panel). (B) Immunofluorescence analysis confirmed the increasing tyrosine phosphorylation of caveolin-1 up to 60 minutes after ephrinB2 ligand stimulation (B2) compared with non-stimulated cells (B1). Thereafter, the phosphorylation of caveolin-1 decreased steadily. CHO-EphB1 cells were fixed and stained with primary antibody against phospho-caveolin followed by Alexa red-conjugated secondary antibody. 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. Bars, 20 µm. (C) CHO cells were serum starved for at least 24 hours, then stimulated for indicated times with 1-2 µg/ml ephrinB2/Fc. Cell lysates were separated by SDS-PAGE, transferred to PVDF membranes, and blotted with antibodies against phospho-caveolin (upper panel) or caveolin-1 (lower panel). Results are representative of at least three independent experiments.





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