DEV02842 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Inhibition of the RhoA-Rho kinase pathway inhibits ephrin-induced COS cell retraction. COS cells were pre-treated with the Rho kinase inhibitor Y-27632 (10 mM) for 30 minutes before stimulation with ephrin-A1 Fc, ephrin-B1 Fc or Fc as a control in the continued presence of the inhibitor. The histogram shows the percentage of cells stained with fluorescently labeled phalloidin that have spikes at the periphery, indicating cell retraction, in a representative experiment.

• Supplemental Figure 2 -

  Fig. S2. Integrin activation by manganese inhibits ephrin-induced COS cell retraction. COS cells were pre-treated with 500 mM manganese for 30 minutes before stimulation with ephrin-A1 Fc, ephrin-B1 Fc or Fc, as a control, in the continued presence of manganese. The histogram shows the percentage of cells stained with fluorescently-labeled phalloidin that have spikes at the periphery, indicating cell retraction, in a representative experiment.

• Supplemental Figure 3 -

  Fig. S3. GTP-bound R-Ras does not inhibit ephrin-induced COS cell retraction if it contains glutamic acid at position 66. COS cells were transiently transfected with EGFP-tagged R-Ras38VY66E or the EGFP vector as a control. The transfected cells were then stimulated with ephrin-A1 Fc, ephrin-B1 Fc or Fc control and stained with fluorescently labeled phalloidin. The histogram shows the percentage of transfected (EGFP-positive) cells that have spikes at the periphery, indicating cell retraction, in a representative experiment. Expression of R-Ras38VY66E, which cannot hydrolyze GTP but contains a negative charge at position 66 as a result of a tyrosine to glutamic acid mutation, does not affect ephrin-induced cell retraction.

• Supplemental Figure 4 -

  Fig. S4. Phosphorylation of Crk does not play a major role in ephrin-induced COS cell retraction and R-Ras inactivation. (A) Ephrin-B1 Fc stimulation increases Crk tyrosine phosphorylation. COS cells treated with either ephrin-B1 Fc or Fc were used for immunoprecipitation with anti-Crk antibodies. Immunoprecipitates were probed with anti-phosphotyrosine antibodies (P-Tyr) and reprobed with anti-Crk antibodies. (B) CrkY221F, a mutant that cannot be inactivated by phosphorylation at tyrosine 221, does not prevent the decrease in R-RasGTP induced by ephrin-B1 Fc treatment. COS cells transiently transfected with a CrkY221F plasmid were stimulated with ephrin-B1 Fc, or Fc as a control, and GTP-bound R-Ras was isolated with GST-Raf1 RBD and detected with anti-R-Ras antibodies. (C) CrkY221F does not prevent cell retraction induced by ephrin Fc treatment. COS cells were transiently transfected with EGFP-tagged CrkY221F or EGFP as a control. Cells were then stimulated with ephrin-A1 Fc, ephrin-B1 Fc or Fc and stained with rhodamine-phalloidin. The histogram shows the mean percentages of transfected cells that have spikes at the periphery and the bars represent standard errors from three experiments.

• Supplemental Figure 5 -

  Fig. S5. Mutation of R-Ras tyrosine 66 to glutamic acid but not phenylalanine completely blocks binding of GTP-bound R-Ras to the Raf-1 RBD. Cells transfected with the indicated R-Ras constructs were incubated with GST-Raf1 RBD and bound proteins and lysates were probed with anti-R-Ras antibodies. Lysates were also probed to verify the amount of the different R-Ras mutants present in the lysates.