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Fig. 6. Stimulation of ephrin-B1 with EphB2 activates Arf1. (A) Flag-tagged ephrin-B1 was expressed in Capan-1 cells. The cells were treated with or without EphB2-Fc (2 µg/ml) and brefeldin A (10 µg/ml) as indicated for 1.5 hours, and conditioned medium was assayed for the 35 kDa ectodomain of ephrin-B1. (B,C) The activity of Arf1 was analyzed in the indicated cells. (C) Wild-type or mutant ephrin-B1 was expressed in Capan-1 cells as in Fig. 2A. The cells were incubated with EphB2-Fc (4 µg/ml) for 20 minutes before being lysed. Arf1-GTP was pulled down with GST-GGA3 bound to glutathione-Sepharose. As controls, lysates of COS-1 cells transiently transfected with plasmids encoding Arf1 T31N or Arf1 Q71L, HA tagged at the C-terminus were analyzed (B, right panel). Open arrowhead indicates cross-reacted GST-GGA3 used for the pull-down assay. (D) Suppression of Arf1 activation decreased the MMP-8 secretion. Capan-1 cells stably expressing ephrin-B1 were used. In the right lane, Arf1 T31N was also transiently expressed in the cells by retrovirus mediated gene transfer. All cells were treated with EphB2-Fc together with (middle lane) or without brefeldin A for 4 hours, and the conditioned medium was subjected to TCA precipitation to detect MMP-8 through immunoblotting. The filled arrowhead indicates endogenous Arf1, and the open arrowhead indicates HA-tagged Arf1 T31N (bottom).
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