Figure 4.
DOCK11 is upregulated in Dcaf8 knockout HSPCs and is polyubiquitinated and destabilized by DCAF8. (A) Venn diagram for proteins that were upregulated in Dcaf8−/− HSPCs or proteins with downregulated ubiquitination in Dcaf8−/− BM cells. (B) Proteins identified in an overlap analysis as shown in panel A. Fold changes are represented as Dcaf8−/− vs WT HSPCs. (C) Immunoblot of DOCK11 in LK cells from WT and Dcaf8−/− mice. (D) Representative immunoblot of DOCK11 and DCAF8 in LT-HSCs from WT and Dcaf8−/− mice; and (E) the relative intensity of DOCK11 immunoblot, quantified and normalized to β-actin (n = 4 independent experiments). (F) RT-qPCR of Dock11 in LT-HSCs from WT and Dcaf8−/− mice (n = 3 technical replicates, pooled from 3 mice). (G) Representative immunoblot of DOCK11 in LT-HSCs at different mouse ages, and (H) the relative intensity of DOCK11 immunoblot, quantified and normalized to β-actin (n = 3 independent experiments). (I-J) Coimmunoprecipitation of DCAF8 and DOCK11. DCAF8 (I) and DOCK11 (J) were immunoprecipitated in cell lysates of Ba/F3 cells, and the associated DCAF8 and DOCK11 were analyzed. (K) HEK293T cells were transiently transfected with the plasmids indicated. Flag-tagged DOCK11 was immunoprecipitated and the ubiquitination was detected with Myc-tagged ubiquitin. (L) HEK293T cells were transiently transfected with WT HA-tagged DCAF8, or DCAF8 mutants R308H or R356H. Flag-tagged DOCK11 was immunoprecipitated and the ubiquitination was detected with Myc-tagged ubiquitin (Ub). (M) Immunoblot of DCAF8 and DOCK11 in Ba/F3 cells stably expressing vectors or Dcaf8 single-guide RNA (sgRNA). Cells were treated with CHX (50 μg/mL) for the indicated durations and lysed. (N) The relative intensity of DOCK11 in panel M was quantified and normalized to β-actin (n = 2 independent experiments). (O) HEK293T cells were transiently transfected with WT Ub or Ub mutants K48R or K63R. Flag-tagged DOCK11 was immunoprecipitated and the ubiquitination was detected with Myc-tagged Ub. Data are presented as mean ± SD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 by unpaired 2-tailed t tests unless otherwise specified. CHX, cycloheximide; FC, fold change; Flag, FLAG tag; HA, hemagglutinin tag; IB, immunoblot; IgG, immunoglobulin G; IP, immunoprecipitation; LK, Lin−c-Kit+; M, months; ns, no statistical significance.

DOCK11 is upregulated in Dcaf8 knockout HSPCs and is polyubiquitinated and destabilized by DCAF8. (A) Venn diagram for proteins that were upregulated in Dcaf8−/− HSPCs or proteins with downregulated ubiquitination in Dcaf8−/− BM cells. (B) Proteins identified in an overlap analysis as shown in panel A. Fold changes are represented as Dcaf8−/− vs WT HSPCs. (C) Immunoblot of DOCK11 in LK cells from WT and Dcaf8−/− mice. (D) Representative immunoblot of DOCK11 and DCAF8 in LT-HSCs from WT and Dcaf8−/− mice; and (E) the relative intensity of DOCK11 immunoblot, quantified and normalized to β-actin (n = 4 independent experiments). (F) RT-qPCR of Dock11 in LT-HSCs from WT and Dcaf8−/− mice (n = 3 technical replicates, pooled from 3 mice). (G) Representative immunoblot of DOCK11 in LT-HSCs at different mouse ages, and (H) the relative intensity of DOCK11 immunoblot, quantified and normalized to β-actin (n = 3 independent experiments). (I-J) Coimmunoprecipitation of DCAF8 and DOCK11. DCAF8 (I) and DOCK11 (J) were immunoprecipitated in cell lysates of Ba/F3 cells, and the associated DCAF8 and DOCK11 were analyzed. (K) HEK293T cells were transiently transfected with the plasmids indicated. Flag-tagged DOCK11 was immunoprecipitated and the ubiquitination was detected with Myc-tagged ubiquitin. (L) HEK293T cells were transiently transfected with WT HA-tagged DCAF8, or DCAF8 mutants R308H or R356H. Flag-tagged DOCK11 was immunoprecipitated and the ubiquitination was detected with Myc-tagged ubiquitin (Ub). (M) Immunoblot of DCAF8 and DOCK11 in Ba/F3 cells stably expressing vectors or Dcaf8 single-guide RNA (sgRNA). Cells were treated with CHX (50 μg/mL) for the indicated durations and lysed. (N) The relative intensity of DOCK11 in panel M was quantified and normalized to β-actin (n = 2 independent experiments). (O) HEK293T cells were transiently transfected with WT Ub or Ub mutants K48R or K63R. Flag-tagged DOCK11 was immunoprecipitated and the ubiquitination was detected with Myc-tagged Ub. Data are presented as mean ± SD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 by unpaired 2-tailed t tests unless otherwise specified. CHX, cycloheximide; FC, fold change; Flag, FLAG tag; HA, hemagglutinin tag; IB, immunoblot; IgG, immunoglobulin G; IP, immunoprecipitation; LK, Lin−c-Kit+; M, months; ns, no statistical significance.

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