Tec is involved in the signaling pathway of GM-CSF receptor. (A) UT-7 cells (1 × 10⁷) were cultured in the starvation medium for 12 hours and then stimulated with 10 ng/mL of human GM-CSF (+) for 5 minutes or left unstimulated (−). Tec was immunoprecipitated from each fraction (αTec), subjected to 7.5% SDS-PAGE, and immunoblotted with anti-phosphotyrosine antibody (αP-Tyr). Total cell lysates (TCL; 10 μg/lane) and the immunoprecipitates by normal rabbit serum (NRS) prepared from the same set of cells were also analyzed. The position of Tec is indicated by an arrow. The molecular weight standards (×10⁻³) are shown at the left. The same membrane was reblotted with anti-Tec serum to show the amounts of Tec precipitated (lower panel). (B) UT-7 cells were stimulated with GM-CSF (10 ng/mL) for 0, 1, 5, 10, or 20 minutes as indicated at the top. Tec was immunoprecipitated from each fraction (1 × 10⁷ cells), and was immunoblotted with anti-phosphotyrosine antibody (αP-Tyr) or anti-Tec serum (αTec). (C) Tec was immunoprecipitated from 1 × 10⁷ of UT-7 cells with (+) or without (−) 5 minutes of GM-CSF stimulation, and was subjected to an in vitro kinase assay. Autophosphorylation of pp70^Tec is shown. (D) Specific kinase activity of the Tec protein (³²P-incorporation/protein amount) with (+) or without (−) the GM-CSF stimulation was calculated by densitometric analysis and shown as arbitrary units. (E) Tec was immunoprecipitated from UT-7 cells (1 × 10⁷), with (GM) or without (−) the GM-CSF stimulation (10 ng/mL), metabolically labeled with [³²P]orthophosphate (37 MBq/mL), and was analyzed by 7.5% SDS-PAGE. The proteins were blotted onto a PVDF membrane, and heated in 1 N KOH to decrease the backgrounds of serine- and threonine-phosphorylation. The position of Tec is indicated. (F) pp70^Tec in (E) was subjected to the phosphoamino acid analysis. The positions of free phosphate (Pi), phosphoserine (p-Ser), phosphothreonine (p-Thr), and phosphotyrosine (p-Tyr) are indicated at the right.