Cloning and characterization of the differentially expressed ckSLAPgene. (A) RT-PCR analyses of SLAP expression. Control and FLI-1-transformed erythroblasts were maintained in the absence or in the presence of Epo as indicated. PCR was carried out with the use of primers for either ckSLAP (top blot) or the gene for ribosomal protein S17 as normalization control (bottom blot). The expected 520-bp SLAP and 129-bp S17 gene fragments are indicated. (B) Northern blot analysis. PolyA+ RNA from control and FLI-1-transformed erythroblasts were isolated from cells incubated for 10 hours in the absence of Epo. Hybridization was carried out with a probe corresponding to clone 50 (top blot) or to ckband4.1 gene as loading control (bottom blot). (C) Western blot analysis. Lysates from control and FLI-1-transformed clones cultivated in Epo for 24 and 48 hours were processed for Western blot by using an antibody to ckSLAP (top blot) or an antibody to ERK as loading control (bottom blot). (D) Alignment of mouse and chicken SLAP sequences. The deduced amino acid sequence of ckSLAP was aligned with that of mSLAP. Identities are shown as vertical lines. The SH3 domain is shaded in light gray and the SH2 domain in dark gray. The sequence corresponding to clone 50 corresponds to the last 144 amino acids. Basic amino acids conserved in the phosphotyrosine binding pocket of SH2 domains and those of the specificity determining region are highlighted by stars and dots, respectively. Hydrophobic residues involved in interactions of SH3 domains with proline-rich ligands are shown as circles. (E) ckSLAP inhibits TCR-induced activation of NFAT. Jurkat cells were cotransfected with 10 μg pΔODLO(NFAT)₃-Luc reporter together with either 5 μg pEFBos control or an increasing amount (0.25, 0.5, 1, or 5 μg) of pEFBos-ckSLAP. Cells were then treated as indicated. Luciferase activity is presented as fold activation with respect to the basal activity of the reporter. The average values and standard deviations of 3 independent experiments are shown.