Lad associates with Gβ in T cells upon chemokine treatment. (A) Establishment of Jurkat T-cell clones that stably express FLAG-tagged full-length Lad (Lad) or Lad-SH2 domain (Lad-SH2). Three independent clones were selected for each transfectant, and the expression of the transfected genes was assayed by Western blotting with anti-FLAG antibody. The positive control (+) is the lysate of Cos7 cells transfected with expression plasmids encoding Lad or Lad-SH2 domain. The negative control (−) is the lysate of untransfected COS7 cells. (B) Lad associates with Gβ in T cells upon chemokine treatment. Jurkat T cells that stably express full-length Lad were activated with anti-CD3ϵ antibody (CD3), SDF-1α (S), or RANTES (R) for 10 minutes. The cell lysates were then subjected to immunoprecipitation with anti-FLAG antibody and Western blotting with anti-Gβ antibody (upper panel). The same precipitates were also analyzed by Western blotting with anti-phosphotyrosine (α-pY) antibody (middle panels) or anti-Flag antibody (lower panel). To visualize the phosphorylated Lad band more clearly, another panel from a shorter exposure of the same blot around the phosphorylated Lad band is shown below middle panel. pLAD indicates phosphorylated Lad; HC, IgG heavy chain