Tec can phosphorylate Jak2 in both mammalian and insect cells. (A) Jak2^KE was immunoprecipitated from 2 × 10⁶ of 293 cells expressing Jak2^KE with (T) or without (−) Tec. Total cell lysates (TCL, 10 μg/lane) and anti-Jak2 immunoprecipitates (Jak IP) were electrophoresed and probed with anti-phosphotyrosine antibody (αP-Tyr) or anti-Jak2 serum (αJak2). The positions of Jak2 (Jak2), Tec (Tec), and the Ig heavy chain (IgH) are indicated at the right. (B) Jak2^KE was immunoprecipitated from Sf21 cells infected with Jak2^KEexpressing baculovirus (J^E) alone or in combination with Tec-expressing (T) or Tec^KM-expressing (T^M) virus. The immunoprecipitates were separated through 7.5% SDS-PAGE and probed with anti-phosphotyrosine antibody (αP-Tyr) or anti-Jak2 serum (αJak2). The positions of Jak2 (Jak2) and Tec (Tec) are indicated at the right. (C) Jak2 was immunoprecipitated from Sf21 cells expressing Jak2 (J) or Jak2^KE (J^E) either alone or in combination with Tec (T). The immunoprecipitates were incubated with [γ-³²P]ATP and the synthetic Jak2-substrate, and subjected to Tricine-SDS-PAGE. Phosphorylation of the Jak2-substrate is shown. (D) Total cell lysates (TCL: 10 μg/lane) and the anti-Jak2 immunoprecipitates (Jak IP) were prepared from parental BA/F3 cells (P) and two BA/F3 clones (1 and 2) stably expressing Tec▵SH3, and immunoblotted with αP-Tyr Ab (upper panel) or anti-Jak2 serum (lower panel). The position of Jak2 is indicated at the right. The positions of molecular weight standards (×10⁻³) are also shown at the left.