Figure 2.

Kinetic analysis of the catalytic activity of Jak2. (A) Expression plasmids for JH1-HA and JH1–2-HA were transfected into 293T cells, and cell lysates were immunoprecipitated using anti-HA antibody. Aliquots of the immunoprecipitates (left) and cell lysates (right) were separated in 4–15% SDS-PAGE and analyzed in an anti-HA immunoblot. The mobilities of the molecular mass markers (in kilodaltons) are shown on the right. (B) Immunoprecipitated JH1-HA and JH1–2-HA were subjected to in vitro kinase assay by using Jak2-derived peptide (1.2 mM) as a substrate. The reactions were stopped at various time points and the peptides were separated in 20% SDS-PAGE followed by quantification using PhosphorImager. (C) Immunoprecipitated JH1-HA and JH1–2-HA were subjected to in vitro kinase assay by using as substrate a range of different Jak2 peptide concentrations, as indicated. The reaction time was 30 min. The peptides were separated in 20% SDS-PAGE followed by quantification with PhosphorImager. (D) Data from C are shown as percentages of maximal JH1 or JH1–2 activities (the values for JH1 are divided by the highest JH1 value, and the values for JH1–2 are divided by the highest JH1–2 value). In B and C, [γ-³³P]ATP was used, and the total concentration of ATP was 250 μM.