Dcaf8 knockout leads to elevated activity of CDC42. (A) Immunoblot of guanosine triphosphate (GTP)-bound (active) CDC42 and total CDC42 in Lin⁻ cells from WT, Dcaf8^−/− mice, and aged (20 months old) WT controls, and (B) the ratio of GTP-bound CDC42 to total CDC42 (n = 3 independent experiments). Statistical analysis used 1-way ANOVA followed by the Tukey multiple comparisons test. (C) Immunoblot of GTP-bound (active) CDC42 and total CDC42 in Ba/F3 cells stably expressing vectors or Dock11 sgRNA. (D) Immunoblot of GTP-bound (active) CDC42 and total CDC42 in Ba/F3 cells stably expressing vectors, overexpressing HA-tagged DCAF8, or expressing Dcaf8 sgRNA, and (E) the ratio of GTP-bound CDC42 to total CDC42 (n = 3 independent experiments). Statistical analysis used 1-way ANOVA followed by the Tukey multiple comparisons test. (F) Representative immunofluorescence images showing the distribution of CDC42 and tubulin in purified LT-HSCs from WT and Dcaf8^−/− mice. Scale bar, 5 μm. (G) Representative intensity plots along sections through locations of distributed CDC42 and tubulin across a cell, as seen in panel F. (H) Percentages of LT-HSCs with polarized distribution of CDC42 and tubulin in WT and Dcaf8^−/− mice (n = 3 independent experiments, in each experiment at least 40 cells pooled from 3 mice were analyzed). Data are presented as mean ± SD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 by unpaired 2-tailed t tests unless otherwise specified. HA, hemagglutinin tag; ns, no statistical significance.