American Society of Hematology

Figure 5.

$Figure 5. Ephrin-A1 induces intracellular tyrosine phosphorylation in CD4+ T cells. (A) CD4+ T cells were stimulated for the indicated times (in minutes) with ephrin-A1-Fc, and subcellular fractionation was performed. The Western blot was incubated with the antiphosphotyrosine antibody PY99. Molecular mass in kDa is shown to the left. Arrows indicate induced phosphorylation. Actin serves as loading control. (B) Incubation of antiphospho-PYK2 antibodies (PYK2 Y580, PYK2 Y403, and PYK2 Y881; Y indicates tyrosine) and total PYK2 antibody to Western blot of subcellular fractionated lysates after ephrin-A1 stimulation. Induced PYK2 phosphorylation is seen on tyrosine residues 580 and 402. (C) PYK2 was immunoprecipitated (IP PYK2) from CD4+ T cells after ephrin-A1 stimulation. Phosphorylated PYK2 was then detected using antiphosphotyrosine antibody PY99 or antiphospho-PYK2 antibodies. Total PYK2 expression serves as control.$

Ephrin-A1 induces intracellular tyrosine phosphorylation in CD4⁺ T cells. (A) CD4⁺ T cells were stimulated for the indicated times (in minutes) with ephrin-A1-Fc, and subcellular fractionation was performed. The Western blot was incubated with the antiphosphotyrosine antibody PY99. Molecular mass in kDa is shown to the left. Arrows indicate induced phosphorylation. Actin serves as loading control. (B) Incubation of antiphospho-PYK2 antibodies (PYK2 Y580, PYK2 Y403, and PYK2 Y881; Y indicates tyrosine) and total PYK2 antibody to Western blot of subcellular fractionated lysates after ephrin-A1 stimulation. Induced PYK2 phosphorylation is seen on tyrosine residues 580 and 402. (C) PYK2 was immunoprecipitated (IP PYK2) from CD4⁺ T cells after ephrin-A1 stimulation. Phosphorylated PYK2 was then detected using antiphosphotyrosine antibody PY99 or antiphospho-PYK2 antibodies. Total PYK2 expression serves as control.

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