FIG. 6.

Socs-1 inhibits kinase activity of TEL-JAK2. (A) TEL-JAK2 in vitro kinase assay. 293T cells expressing 5/19 TEL-JAK2 and increasing amounts of Myc–Socs-1 were starved of FBS for 4 h. Extracts from these cells were immunoprecipitated (IP) with a TEL antibody and used for in vitro kinase assays using GST-C′Gab2 as a substrate. Products were separated by SDS-PAGE and visualized by autoradiography (top). Parallel TEL Western blotting confirmed equal immunoprecipitation of the protein (bottom). (B) 4G10 Western blot. 293T cells expressing 5/19 TEL-JAK2 and increasing amounts of Myc-Socs-1 were starved of FBS for 4 h. TEL-JAK2 was immunoprecipitated with the JAK2 antibody and blotted with 4G10 (top) or JAK2 antibody (bottom). The whole-cell lysates were blotted with a Myc antibody to confirm expression of Socs-1 and a TEL antibody to confirm equal expression of TEL-JAK2 (data not shown). (C) Phosphoamino acid analysis of C′Gab2. Phosphorylated GST-C′Gab2 substrate from panel was hydrolyzed to single amino acids and separated on a thin-layer chromatography plate (left). The plate was placed on a phosphorimager, and the ratio of phosphorylated amino acids was used to generate the graph (right). Open bars represent TEL-JAK2 samples, and solid bars represent Socs-1 and TEL-JAK2 samples. (D) Phosphoamino acid analysis of autophosphorylated TEL-JAK2. Analysis of the autophosphorylated TEL-JAK2 was performed as for panel C. Open bars represent TEL-JAK2 samples, and solid bars represent Socs-1 and TEL-JAK2 samples.