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. 2005 May 2;201(9):1421–1433. doi: 10.1084/jem.20042294

Figure 6.

Figure 6.

Requirement of CD45 in Ly49D-dependent Syk activation in NK cells. IL-15–expanded CD45 − / − and control NK cells were starved of cytokine and serum for 2 h and then stimulated with anti-Ly49D mAb at 37°C for the indicated times. (A) Whole cell lysates were probed with anti-phosphotyrosine or with protein loading verified using anti–β-actin. (B) Syk or (C) Zap70 were immunoprecipitated from Ly49D-stimulated CD45 − / − and control NK cell lysates and probed for antiphosphotyrosine and anti-Syk or anti-Zap70 to determine protein loading. (D) IL-15–expanded CD45 − / − and control NK cells were loaded with Indo-1-AM and stained with biotinylated anti-Ly49D. The ratio of fluorescence detected in FL5–FL4 was monitored by flow cytometry for 5 min with the addition of avidin to cross-link Ly49D receptors at 30 s. Data are a representative of at least two independent experiments. (E) Indo-1-AM–loaded NK cells were treated with a calcium ionophore at 30 s. Changes in fluorescence were monitored for 4 min. Data are a representative of at least two independent experiments.