SLAP interferes with Epo-induced differentiation of EI/11 erythroblasts. (A) Expression of exogenous SLAP protein in EI/11-eGFP/SLAP culture was analyzed by Western blotting using an anti-Flag antibody. (B) Hemoglobin content in control eGFP and eGFP/SLAP cells maintained under proliferation conditions (day 0) and after differentiation induction for 1, 2, 3, and 4 days in the presence of Epo. Values were normalized with respect to cell number and volume (hemoglobin level per 10⁶ live cells). The mean value and standard deviation of triplicate measurements are shown. (C) Quantitative evaluation of apoptosis by TUNEL assay in eGFP and eGFP/SLAP cells maintained in differentiation conditions for 48 or 72 hours. (D) RT-PCR analysis of BCL-X_L mRNA expression in control eGFP and eGFP/SLAP erythroblasts maintained under either proliferative conditions (0 hours) or induced to differentiate for 48 hours in the presence of Epo. The mean value and standard deviation of 3 independent fields are shown. (D) PCR was carried out using 0.5 μL and 1 μL RT reactions. Primers used were specific for either the BCL-X_L gene (top blot) or the β-Actin gene as normalization control (bottom blot). The expected 610-bp BCL-xL fragment and 540-bp Actin gene fragment are indicated.