Fig. 2.

Growth factor withdrawal induces phosphorylation/activation of MKK3/6, p38α, and MAPKAP-2 in primary erythroid progenitors. Primary erythroid progenitors on day 8 of culture were deprived of Epo and SCF for indicated periods or stimulated with Epo or SCF after growth factor starvation. Cell lysates were collected and analyzed by immunoblotting with anti-phospho MKK3/6 antibodies (A) or anti-phospho p38α antibodies (B). Each of the blots were then stripped and reprobed with either anti-tubulin (A) or anti-p38α (B) antibodies to confirm equal protein loading. (C) Day 8 cells were growth factor starved as indicated and stimulated with Epo or SCF. Total cell lysates were immunoprecipitated with antibodies against MAPKAP-2. The immunocomplexes were then used in kinase assays. Hsp25 protein was used as a substrate along with [γ-³²P]ATP in the reactions. Subsequently, proteins were analyzed by SDS/PAGE, and the phosphorylated form of Hsp25 was detected by autoradiography.