The EGF receptor/EPO receptor chimera EECA-Y429, 431F mediates SKT6 cell hemoglobinization at enhanced levels (versus the control chimeric construct EECA). (A) Levels of expression of EECA and EECA-Y429, 431F receptor forms in stably transfected SKT6 cell lines were assayed by Northern blotting. Hybridization was performed using a 1,700-bp EcoRI → Bgl II cDNA fragment of ECCA (see the Materials and Methods). (B) SKT6 cells were electrotransfected with pCINeo vectors encoding the chimeric receptors EECA or EECA-Y429, 431F, and stably transfected cell lines (SKT6-EECA and SKT6-EECA-Y429, 431F ) were isolated by culture in G418 (see the Materials and Methods). In these derived lines, the ability of these chimeric receptors to mediate EGF-induced hemoglobinization then was compared. Briefly, cells (2.5 × 10⁵ cells/mL) were exposed to EGF (±25 ng/mL) or EPO (±2 U/mL) for 72 hours and the frequencies of ligand-induced hemoglobinization were assayed by in situ staining with DAF. Values are means ± standard deviations for three replicate cultures and are corrected for background (ie, low levels of DAF-positive cells observed in the absence of EGF or EPO are subtracted). Two clones from each electrotransfection are represented. (C) To provide for a more direct comparison of the activities of EECA and EECA-Y429, 431DF receptor forms, data from above (A) for SKT6-EECA and SKT6-EECA-Y429, 431DF cell lines are compared directly with frequencies of hemoglobinization in these lines as induced by EPO in parallel cultures. (D) To confirm results obtained via the staining of hemoglobinized SKT6 cells with DAF, levels of globin in lysates from the above cultures also were assayed by Western blotting.