FIG. 3.

Stimulation of gp130 but not d771-811 induces activation of Hck in the growth factor-dependent cell line Baf-B03. Baf-B03 cells were transfected with expression vectors for Hck (pDpuro Hck) and for Eg and d771-811, respectively (pcDNA6/V5-His). These cells were then either stimulated with 40 U of EPO/ml (+) or with medium only (−). The activity of Hck was tested by performing in vitro kinase assays in the presence of radiolabeled ATP and the external Src kinase substrate Sam 68 as described in the text. After separation with SDS–10% PAGE the EPO-induced phosphorylation of Sam 68 was assessed by autoradiography (upper panel). Aliquots of the Hck immunoprecipitate were blotted with anti-Hck antibody (α-Hck) to control for comparable precipitation. The results were quantified by using phosphorimaging analysis software. Normalized induction levels of kinase activity were as indicated.