Tyrosine phosphorylation of STAT3 and MAPK in BAF/GCSFR cells and human neutrophils. (A) BAF/GCSFR cells were treated for 15 minutes at 37°C with the small compounds (10^–6 M) or rhG-CSF (6 × 10^–10 M). Cell lysates were separated by SDS-PAGE and examined with antibodies against STAT3, phospho-STAT3, MAPK, or phospho-MAPK. (B) Phosphorylation of STAT3 and MAPK in human neutrophils. Purified human neutrophils were incubated for 20 minutes at 37°C with small compounds (10^–6 M) or rhG-CSF (6 × 10^–10 M). Proteins were immunoprecipitated from the cell lysates with an antibody against STAT3. The immunoprecipitated proteins were separated by SDS-PAGE and detected with an antibody against phospho-STAT3. Expression of STAT3 was detected with an antibody specific to this protein. Cell lysates were separated on SDS-PAGE and the gels stained with antibodies to phospho-MAPK and MAPK. Results are representative of 3 separate experiments.