Fig. 3. IEX-1 binds specifically to the active forms of ERK1/2. (A and B) Sepharose-bound GST or GST–IEX-1 wild type and mutants were incubated with lysates from UT7 cells either untreated (0) or stimulated with 10 nM TPO or 100 ng/ml anisomycin (Aniso) for 30 min, as indicated. MAPKs were detected in GST precipitates or in samples of total lysates (TL) by immunoblotting with antibodies directed against the active forms of ERK, JNK or p38. (C and D) Cos7 cells were transfected with 2 µg of pcDNA-HA-IEX-1 (Wt or ΔBD mutant) or empty vector (V), starved overnight and stimulated (+) or not (–) with 100 ng/ml EGF for 10 min. Lysates were immunoprecipitated (IP) with anti-HA or anti-ERK1 antibodies and analyzed by western blotting. Expressions of ERK and HA-IEX-1 are shown in total lysates (TL). (E) UT7 cells (50 × 10⁷) were treated with TPO for 3 h to induce endogenous IEX-1 protein expression. The presence of IEX-1 and phosphorylated ERK was monitored in anti-IEX-1, anti-phosphoERK or control (C) immunoprecipitates (IP), as indicated. As a control, phosphoERK and IEX-1 expressions are shown in total lysates (TL) from 1 × 10⁶ cells.