SH2 domain of Lnk interacts with MPLWT and MPLW515L. (A-C) Bone marrow cells (A) from Lnk^+/+ and Lnk^−/− mice (6 each) were cultured with Tpo (50 ng/mL, 3 days). UT7/MPL515 cells (B) were cultured either without or with Tpo (1 ng/mL). 293T cells (C) were transfected with combinations of MPLWT, MPLW515L, wild-type Lnk (LW), or the SH2 mutant Lnk (LM) as indicated and were either untreated or treated with Tpo (1 ng/mL, 30 minutes). Lysates were immunoprecipitated (IP) with MPL or Lnk antibodies and analyzed by Western blot as indicated. Total Lnk and MPL levels were analyzed by Western blot (bottom panels). (D) Protein lysates from 293T cells transfected with either MPLWT or MPLW515L were incubated with either GST protein or GST-Lnk-SH2 fusion protein (GST-L-SH2). GST-protein complexes were analyzed by Western blot with MPL antibody. Lysates also were immunoprecipitated with a phosphotyrosine antibody and analyzed by Western blot with MPL antibody (right panel). (E) 293T cells were transfected and treated as described in panel C. Lysates were immunoprecipitated with phosphotyrosine antibody and analyzed by Western blot with Lnk antibody (top panel). Lnk levels in the lysates were analyzed by Western blot (bottom panel).