FIGURE 1.

Hck is required for tyrosine phosphorylation of WASP. A, wild-type and Hck^−/− BMMs were untreated (− or none) or stimulated with either CX3CL1 for 1 min (+) or with EIgG for 5min. WASP was immunoprecipitated using WASP antibody, followed by Western blotting with either HRP-conjugated phospho-tyrosine (PY) and WASP antibodies. Blots were quantified by densitometry and normalized to the amount of immunoprecipitated WASP. Data are expressed as the fold increase as compared with WT prior to stimulation. ± S.E. (n = 3). *, p < 0.05. B, whole cell lysates of the same conditions were also probed with HRP conjugated phospho-tyrosine. C, RAW/LR5 cells were transfected with control shRNA (Ctrlsh) or two different Hck shRNA (Hcksh1 and Hcksh2) plasmids and selected for 2 days in puromycin. Hck and β-actin expression in these cells was analyzed by Western blotting with the respective antibodies and quantified as the percentage of Hck/β-actin signal intensity ratios relative to the control. D, control and Hck shRNA cells were stimulated with CX3CL1 for 1 min, and then WASP was immunoprecipitated using WASP antibody, followed by Western blotting with the HRP-conjugated phospho-tyrosine (PY) and WASP antibody. Data are expressed as the fold increase as compared with non-stimulated control shRNA cells. ± S.E. (n = 3), **, p < 0.01 compared with CX3CL1-induced tyrosine phosphorylation in control shRNA cells.