MS-1020 inhibits JAK3 kinase activity in vitro. JAK2 (A) or JAK3 (B) immunoprecipitates were used for in vitro kinase assay. Kinase reactions were performed by the addition of recombinant His-tagged STAT3α protein as a substrate, 2 μmol/L ATP (20 μmol/L ATP for competition experiments, ++), and MS-1020 at different concentrations or the JAK kinase inhibitor AG490 (150 μmol/L). The reaction products were resolved on SDS-PAGE and probed with antibodies specific for phospho-STAT3, STAT3, JAK2, and JAK3. Note that MS-1020 selectively blocks JAK3 kinase activity and that MS-1020 is an ATP-competitive JAK3 inhibitor. His-tagged STAT3α, JAK2, and JAK3 serve as a loading control.