SLAP interferes with STAT5 activation. (A) Electrophoretic mobility shift assay analysis. (Top blot) Cell extracts were tested for STAT5 DNA binding by using a STAT5-specific [³²P]-labeled probe. (Bottom blot) The same extracts were incubated with a [³²P]-labeled ETS probe, as loading control. The STAT5-containing specific complex is indicated by an arrow. The arrowhead points to a nonspecific complex. (B) Phosphorylation of STAT5 (i) and ERK (iii) was analyzed by Western blotting with the use of a STAT5 A/B phosphotyrosine-specific antibody or a phospho-ERK antibody. Immunoblotting with antibodies to STAT5 (ii) and ERK2 (iv) was used to quantify the total amount of STAT5 and ERK. (C) SLAP interferes with STAT5 phosphorylation during differentiation. Control eGFP and eGFP/SLAP EI-11 erythroblasts were induced to differentiate in response to Epo. Lysates were collected at 0, 24, 48, and 72 hours and analyzed by Western blot using the STAT5 A/B phosphotyrosine-specific antibody (top blot) or a STAT5 antibody to assess the total amount of STAT5 (bottom blot).