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Fig. 8. EphB activation by ephrin-B1–Fc increases RhoA activity and decreases Rac1 activity, and the RhoA kinase inhibitor Y27632 antagonizes the effects of ephrin-B1–Fc in primary cultures of medullary tubule cells. (A) In cells stimulated for 15 minutes with 1 µg/ml ephrin-B1–Fc, or 1 µg/ml Fc as a control, activated RhoA was isolated by pull-down with a GST fusion protein of the Rho-binding domain of Rhotekin (GST-RBD). Proteins bound to GST-Rhotekin were separated by SDS-PAGE and probed by immunoblotting with an anti-RhoA antibody. The levels of active RhoA in ephrin-B1–Fc-treated cells were normalized to those in Fc control-treated cells. Data from three independent experiments are shown as mean ± s.d. Ephrin-B1 Fc treatment increased the level of activated RhoA by 1.7 fold (P=0.002). (B,C) Phase-contrast time-lapse microscopy photographs. Cells were stimulated with 1 µg/ml ephrin-B1–Fc in the presence of 10 µM Y27632 and a series of phase-contrast images of the same field were obtained at the indicated times. Y27632 was added 10 minutes before (B) and 20 minutes after (C) the addition of ephrin-B1–Fc. Pretreatment with the Y27632 inhibitor essentially abolished the effects of ephrin-B1–Fc (B). Treatment with the inhibitor after cell retraction has begun reverses the effects within 5 minutes (B, arrowheads). (D) Quantification of the percentage of retracting cells stimulated with 1 µg/ml ephrin-B1–Fc under the presence of 10 µM Y27632. Y27632 and ephrin-B1–Fc are added at the same time courses as B and C. Phase-contrast images of the same field were obtained: for the Y27632 addition before the ephrin-B1–Fc stimulation at 0 minute, 10 minutes (after the addition of Y27632) and 30 minutes (20 minutes after the stimulation with ephrin-B1–Fc); for the Y27632 addition after the stimulation at 0 minute, 20 minutes (after the stimulation with ephrin-B1–Fc), and 30 minutes (10 minutes after the addition of Y27632). The fraction of retracting cells at the 10 and 30 minutes time point (open column), and 20 and 30 minutes (dotted column) was calculated, respectively. Only cells at the periphery of the epithelial sheets were quantitated for statistical analysis. Data from three independent experiments are shown as mean ± s.d. (E) Focal adhesion rearrangements induced by treatment with ephrin-B1–Fc are blocked by pretreatment with the Y27632 Rho kinase inhibitor. Medullary tubule cells plated on Matrigel were preincubated with 10 µM Y27632 for 5 minutes and then stimulated for 15 minutes with 1 µg/ml ephrin-B1–Fc, or human Fc as a control. The cells were then stained with an anti-vinculin antibody. (F) In cells stimulated for 5 minutes and 15 minutes with 1 µg/ml ephrin-B1–Fc, or for 15 minutes with 1 µg/ml Fc as a control, activated Rac1 was isolated by pull-down with a GST fusion protein of the Rho-binding domain of PAK1 (GST-PBD). Proteins bound to GST-PAK1 were separated by SDS-PAGE and probed by immunoblotting with an anti-Rac1 antibody. The levels of active Rac1 in ephrin-B1–Fc-treated cells were normalized to those in Fc control-treated cells. Data from three independent experiments are shown as mean ± s.d.
