FIGURE 6.

Compromised Rac2 activation and chemotaxis in Dock2 shRNA cell lines. Polyclonal HL60-CXCR2 cells stably expressing Dock2 shRNA were constructed by lentiviral transfection followed by selection with 0.5 μg/ml puromycin. A, protein expression of Dock2 in dHL60 cells was detected with Western blot. Tubulin was used as loading control. NS is non-silencing shRNA control. 889 and 893 are two individual Dock2 shRNAs. B, Rac2 activation was detected by GST-PBD pull-down assay in DMSO (control) or wortmannin (50 nm, 30 min)-pretreated dHL60 cells. Quantification (right panel) from three independent experiments is shown in the right panel.^*, p < 0.05 and ^**, p < 0.01 (one-way ANOVA with Bonferoni post-tests). C, chemotaxis assay performed in microfluidic devices. The movement of 15–20 randomly picked cells was tracked with the Metamorph program. The direction of the CXCL8 gradient is shown by the arrow in each panel.