Inhibition of cytokine-induced activation of MAPK and Akt by kinase inhibitors.

(A) Inhibition of phosphorylation of MAPK and Akt by kinase inhibitors. JYTF-1 cells were cytokine starved for 24 hours before treatment with PD98059 (lanes 4-6) or LY294002 (lanes 7-9). After 1-hour pretreatment with drugs, cytokines were added to the culture media for 5 minutes before cells were harvested. Cell lysates were prepared and subjected to Western blot analysis for the activation of MAPK and Akt as described in Figure 4. DMSO was used as the solvent and was included as a negative control (lanes 1-3). The same experiment was repeated twice and a representative result is shown here. (B) Inhibition of kinase activities of MAPK and Akt by inhibitors. JYTF-1 cells were treated as described in panel A and cell lysates were immunoprecipitated with anti-P-MAPK or Akt antibody. The immunoprecipitates were then subjected to in vitro kinase assay for both MAPK and Akt, as described in “Materials and methods.” The results of MAPK activity were visualized using an ECL system and the results of Akt activity are shown as an autoradiograph. P-Elk1 is the product of the kinase reaction of MAP kinase and H2B is the substrate of Akt kinase.