Figure 4.

LKB1 is tyrosine phosphorylated following TCR signal stimulation. (A) Sorted DP thymocytes were treated with or without the Lck inhibitor (5 μM) and stimulated as described above. LKB1 was subsequently precipitated by anti-LKB1 antibodies. The immunoprecipitated LKB1 protein was analysed for tyrosine phosphorylation levels and precipitated LKB1 served as a loading control. (B) 293T cells were transfected with plasmids encoding LKB1 alone or co-transfected with Lck Y505F and harvested 36 h later. Lysates were precipitated with anti-Flag beads and immunoprecipitated proteins were immunoblotted with the indicated antibodies. The asterisk indicates the IgG heavy chain. (C) Thymocyte lysates were incubated with control IgG or anti-LKB1 antibodies and precipitated with protein A/G-agarose beads. The immunoprecipitated proteins were immunoblotted using the indicated antibodies. (D) 293T cells were transfected with plasmids encoding LKB1 alone or co-transfected with Lck or Lck Y505F and harvested for 36 h. Lysates were precipitated with anti-Flag beads and the immunoprecipitated proteins immunoblotted with the indicated antibodies. The asterisks indicate the IgG heavy chain. (E) His-tagged LKB1 was used for the in vitro kinase assay with His-tagged Lck Y505F. Purified His-tagged LKB1 was incubated with or without purified His-tagged Lck Y505F in Src kinase assay buffer. Tyrosine phosphorylation levels of LKB1 were analysed by immunobloting. All results are representative of three independent experiments.