American Society of Hematology

Figure 6.

$Figure 6. Effect of Tyrphostin-A9 and SU6656 on ephrin-A1-induced chemotaxis and intracellular signaling. (A) Effect of Tyrphostin-A9 on ephrin-A1-induced SDF-1α chemotaxis. Fc indicates control Fc; A1, ephrin-A1-Fc. Peripheral blood CD4+ cells were preincubated with the indicated concentrations of Tyrphostin-A9 (in μM) shown in parentheses. SDF-1α was added to the lower chamber and Fc and A1 to the transwell. SEM is shown for 4 different experiments. (B) Effect of Tyrphostin-A9 on ephrin-A1-induced intracellular tyrosine phosphorylation. CD4+ T cells were preincubated with or without 10 μM Tyrphostin-A9 (Tyr A9) and then stimulated for the indicated times (in minutes) with ephrin-A1-Fc. The Western blot of the cytosol + plasma membrane fraction was incubated with the antiphosphotyrosine antibody PY99. Molecular mass in kDa is shown to the left. Actin detection serves as loading control. (C) Effect of Tyrphostin-A9 on PYK2- and EphA1-induced tyrosine phosphorylation. CD4+ T cells were preincubated or not with 10 μM Tyrphostin-A9 (Tyr A9) before stimulation with ephrin-A1-Fc for the indicated time points (in minutes). Western blot was incubated with antiphospho-PYK2 Y580 antibody. Total PYK2 detection serves as loading control. EphA1 was immunoprecipitated (IP) from cell lysates before SDS-PAGE. Phosphorylation of EphA1 was detected with the PY99 antibody. The blot was then stripped and EphA1 levels were confirmed with an anti-EphA1 antibody. (D) Effect of src-family kinase inhibitor SU6656 on ephrin-A1-induced SDF-1α chemotaxis. Fc indicates control Fc; and A1, ephrin-A1-Fc. CD4+ cells were preincubated with the indicated concentrations of SU6656 (in μM) shown in parentheses. SDF-1α was added to the lower chamber and Fc and A1 to the transwell. SEM is shown for 4 different experiments. (E) Effect of SU6656 on ephrin-A1-induced intracellular tyrosine phosphorylation. CD4+ T cells, preincubated or not with 10 μM SU6656, were stimulated for the indicated times (in minutes) with ephrin-A1-Fc. Western blot of cytosol + plasma membrane fraction was incubated with the antiphosphotyrosine antibody PY99. Molecular mass in kDa is shown to the left. Actin detection serves as loading control. (F) Effect of SU6656 on ephrin-A1-induced tyrosine phosphorylation of PYK2. CD4+ T cells were preincubated or not with 10 μM SU6656 and lysates were subcellular fractionated. Western blot was incubated with antiphospho-PYK2 Y580 antibody. Total PYK2 detection serves as loading control.$

Effect of Tyrphostin-A9 and SU6656 on ephrin-A1-induced chemotaxis and intracellular signaling. (A) Effect of Tyrphostin-A9 on ephrin-A1-induced SDF-1α chemotaxis. Fc indicates control Fc; A1, ephrin-A1-Fc. Peripheral blood CD4⁺ cells were preincubated with the indicated concentrations of Tyrphostin-A9 (in μM) shown in parentheses. SDF-1α was added to the lower chamber and Fc and A1 to the transwell. SEM is shown for 4 different experiments. (B) Effect of Tyrphostin-A9 on ephrin-A1-induced intracellular tyrosine phosphorylation. CD4⁺ T cells were preincubated with or without 10 μM Tyrphostin-A9 (Tyr A9) and then stimulated for the indicated times (in minutes) with ephrin-A1-Fc. The Western blot of the cytosol + plasma membrane fraction was incubated with the antiphosphotyrosine antibody PY99. Molecular mass in kDa is shown to the left. Actin detection serves as loading control. (C) Effect of Tyrphostin-A9 on PYK2- and EphA1-induced tyrosine phosphorylation. CD4⁺ T cells were preincubated or not with 10 μM Tyrphostin-A9 (Tyr A9) before stimulation with ephrin-A1-Fc for the indicated time points (in minutes). Western blot was incubated with antiphospho-PYK2 Y580 antibody. Total PYK2 detection serves as loading control. EphA1 was immunoprecipitated (IP) from cell lysates before SDS-PAGE. Phosphorylation of EphA1 was detected with the PY99 antibody. The blot was then stripped and EphA1 levels were confirmed with an anti-EphA1 antibody. (D) Effect of src-family kinase inhibitor SU6656 on ephrin-A1-induced SDF-1α chemotaxis. Fc indicates control Fc; and A1, ephrin-A1-Fc. CD4⁺ cells were preincubated with the indicated concentrations of SU6656 (in μM) shown in parentheses. SDF-1α was added to the lower chamber and Fc and A1 to the transwell. SEM is shown for 4 different experiments. (E) Effect of SU6656 on ephrin-A1-induced intracellular tyrosine phosphorylation. CD4⁺ T cells, preincubated or not with 10 μM SU6656, were stimulated for the indicated times (in minutes) with ephrin-A1-Fc. Western blot of cytosol + plasma membrane fraction was incubated with the antiphosphotyrosine antibody PY99. Molecular mass in kDa is shown to the left. Actin detection serves as loading control. (F) Effect of SU6656 on ephrin-A1-induced tyrosine phosphorylation of PYK2. CD4⁺ T cells were preincubated or not with 10 μM SU6656 and lysates were subcellular fractionated. Western blot was incubated with antiphospho-PYK2 Y⁵⁸⁰ antibody. Total PYK2 detection serves as loading control.

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