Figure 4.

ATA inhibits JAK1 and JAK2 autophosphorylation. RAW 264.7 cells were pretreated with ATA at the concentrations indicated for 20 min followed by the stimulation with IFN-γ (3 ng ml⁻¹) for 10 min. Whole cell lysates were immunoprecipitated with JAK1 or JAK2 antibody, and protein A/G beads overnight. One half of the immunoprecipitated JAK complex was subjected to SDS–PAGE, rendered for the immunoblotting, and probed with antibody specific for JAK. The other half of the immunoprecipitated JAK complex was rendered for kinase assay. After 30 min reaction at room temperature, the enzyme activity was terminated by adding SDS sample buffer and analysed by autoradiography. The results are representative of three different experiments with similar results.