FIG. 5.

Socs-1 inhibits association of Shc and Grb2 with TEL-JAK2 and activation of Erk2. (A) Socs-1 inhibits tyrosine phosphorylation of Shc and association with TEL-JAK2. Extracts from 293T cells expressing 5/19 TEL-JAK2 and increasing amounts of Myc–Socs-1 were immunoprecipitated (IP) with a Shc antibody and blotted with 4G10 (top) or a Shc antibody (bottom). TEL-JAK2 is seen as a doublet due to an alternative start site for translation in TEL. 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; Fig. 6A). (B) Socs-1 inhibits association of GRB2 with TEL-JAK2. Extracts from panel A were immunoprecipitated with a Grb2 antibody and blotted with a TEL antibody (top) or a Grb2 antibody (bottom). (C) Socs-1 inhibits activation of Erk2 in cells expressing TEL-JAK2. 293T cells expressing equivalent amounts of 5/19 TEL-JAK2 and increasing amounts of Myc–Socs-1 were starved of FBS for 4 h and stimulated with 10% FBS for 5 min where indicated. Erk2 immunoprecipitated from these extracts was used for in vitro kinase assays using GST–Elk-1 as a substrate (top). Products were separated by SDS-PAGE followed by autoradiography. Parallel Erk2 Western blotting confirmed immunoprecipitation of the protein (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; Fig. 6A).