Constitutive SLAP expression interferes with Epo-induced differentiation. (A) Expression of exogenous proteins in EpoR and EpoR/SLAP erythroblast clones was analyzed by Western blotting by using either an anti-EpoR (left panel, top blot) or the anti-ckSLAP antibody (right panel, top blot). Expression of endogenous SLAP in a representative EpoR/FLI-1 erythroblast clone is shown in lane 2, right panel. (Bottom blots) ERK expression is shown as a loading control. (B) Quantitative determination of hemoglobin content in 2 EpoR and 2 EpoR/ckSLAP representative clones at days 1, 2, and 3 after differentiation induction in response to Epo. Normalized values (hemoglobin level per 106 live cells) are shown. (C) Cytocentrifugation analysis of EpoR and EpoR/ckSLAP representative clones maintained in absence (-) or presence (+) of Epo for 1, 2, and 3 days. Cells were stained with neutral benzidine (stains hemoglobin in brown) and histologic dyes (Diff-Quik). Original magnification × 60.
