Recombinant IL-7 enhanced tyrosine phosphorylation of proteins in HS-27a.

In panels A and B, tyrosine phosphorylated proteins were analyzed by immunoblots using an antiphosphotyrosine antibody. (A) HS-27a cells were cultured for 5 minutes in standard media (lane 1), or media containing 2 ng/mL IL-1β (lane 2), 10 ng/mL IL-7 (lane 3), or 100 ng/mL IL-7 (lane 4). (B) Cells were cultured in the presence of 100 ng/mL IL-7 for various times as shown. Arrows indicate positions of the tyrosine phosphorylated proteins, which increased after exposure to recombinant IL-7. MW shows a lane of molecular weight marker proteins. (C) Tyrosine-phosphorylated proteins were immunoprecipitated with the biotinylated recombinant RC20 antiphosphotyrosine antibody and immunoblotted with the anti–IL7R-IC antibodies. Cells were cultured in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of 100 ng/mL IL-7 for 1 hour. Total proteins of the cells were extracted under denaturing conditions and immunoprecipitated with (lanes 1 and 3) or without (lanes 2 and4) the RC20 antibody. Mock immunoprecipitation was conducted with the RC20 antibody but without cellular extracts (lane 5).