SDF1α-induced HSPC directional migration and velocity are regulated by the kinase activity of Pak2 and its interaction with β-Pix. WT and Pak2^fl/fl LSK cells were transduced with indicated retroviruses, and FACS-sorted EGFP⁺ cells were imaged using a time-lapse microscope at 30-second intervals for 1 hour after the addition of SDF1α. The cells were tracked as described in “Methods” and analyzed using Metamorph software to generate tracks of migration for each individual cell and for quantification. (A) HSPC migration in response to SDF1α. Origins of all tracks were centered at the 0,0 XY coordinate with distance in micrometers on x- and y-axes shown (n > 11 per group). (B) Schematic representation of migration analysis; (C) the Euclidean distance in micrometers; (D) the velocity of migration in micrometers per second; and (E) the accumulated distance in micrometers. All data expressed as mean ± standard error of the mean (SEM). In panel E, only Pak2-Δβ-Pix is significantly different from control. One of the 3 independent experiments shown and the statistical significance was determined using the Mann-Whitney U test. *P < .05. n.s., not significant.