Figure 5.
Figure 5. DOCK7 interacts with p116Rip. (A) DOCK7 and p116Rip domain structure. (Top) DOCK7 fragments used in YTH screen/testing. CC, coiled-coil domain; PH, pleckstrin homology domain. (B) YTH interaction between DOCK7-R2 and p116Rip. Yeast transformed with plasmids expressing DOCK7-R2 or DOCK7-R3 fused to GAL4 DNA–binding domain (GBD) and p116Rip-C (amino acids 505–933) fused to the GAL4-activation domain (GAD) were grown on medium lacking histidine. Lamin served as negative and H-Ras and phosphatidylinositol 3-OH–kinase-δ (PI3KD) as positive controls. (C) Expression of p116Rip in DCX-positive neuroblasts along the V-SVZ–RMS–OB pathway. Coronal sections of P10 mouse forebrain at positions indicated in the cartoon in Fig. 1 A immunostained with antibodies to p116Rip and DCX and counterstained with DAPI. (D) DOCK7–p116Rip interaction in mammalian cells. Lysates from HEK293 cells transiently expressing Flag-DOCK7 or empty control vector were immunoprecipitated (IP) with an antibody to Flag and analyzed by immunoblotting with antibodies to Flag and p116Rip. TL, total lysate. (E) GST-p116Rip-C (amino acids 505–1,024) fusion protein or GST alone (bottom, Coomassie brilliant blue [CBB] staining), immobilized on beads, was incubated with lysates from P5 mouse brains. Bound DOCK7 was detected by immunoblotting with an antibody to DOCK7. (F) Interaction of DOCK7 and p116Rip in the brain. P10 mouse whole-brain extracts were immunoprecipitated with normal rabbit IgG, an anti-DOCK7 (DOCK7-Ab), or an anti-p116Rip (p116Rip-Ab) antibody and then were analyzed by immunoblotting with antibodies to DOCK7 and p116Rip. Molecular masses are shown in kilodaltons. (G and H) Colocalization of DOCK7 and p116Rip in V-SVZ–derived neuroblasts. (G) Coronal sections of P10 mouse forebrain at position III in the RMS as indicated in the cartoon in Fig. 1 A immunostained with antibodies to DOCK7 and p116Rip and counterstained with DAPI. Boxed regions are enlarged and shown at the bottom. (H) Neuroblasts dissociated from V-SVZ tissue of P1–3 mice were fixed at 30 h in vitro, followed by immunostaining with indicated antibodies and counterstained with DAPI. Bars: (C and G, top) 50 µm; (G, bottom, and H) 10 µm.

DOCK7 interacts with p116Rip. (A) DOCK7 and p116Rip domain structure. (Top) DOCK7 fragments used in YTH screen/testing. CC, coiled-coil domain; PH, pleckstrin homology domain. (B) YTH interaction between DOCK7-R2 and p116Rip. Yeast transformed with plasmids expressing DOCK7-R2 or DOCK7-R3 fused to GAL4 DNA–binding domain (GBD) and p116Rip-C (amino acids 505–933) fused to the GAL4-activation domain (GAD) were grown on medium lacking histidine. Lamin served as negative and H-Ras and phosphatidylinositol 3-OH–kinase-δ (PI3KD) as positive controls. (C) Expression of p116Rip in DCX-positive neuroblasts along the V-SVZ–RMS–OB pathway. Coronal sections of P10 mouse forebrain at positions indicated in the cartoon in Fig. 1 A immunostained with antibodies to p116Rip and DCX and counterstained with DAPI. (D) DOCK7–p116Rip interaction in mammalian cells. Lysates from HEK293 cells transiently expressing Flag-DOCK7 or empty control vector were immunoprecipitated (IP) with an antibody to Flag and analyzed by immunoblotting with antibodies to Flag and p116Rip. TL, total lysate. (E) GST-p116Rip-C (amino acids 505–1,024) fusion protein or GST alone (bottom, Coomassie brilliant blue [CBB] staining), immobilized on beads, was incubated with lysates from P5 mouse brains. Bound DOCK7 was detected by immunoblotting with an antibody to DOCK7. (F) Interaction of DOCK7 and p116Rip in the brain. P10 mouse whole-brain extracts were immunoprecipitated with normal rabbit IgG, an anti-DOCK7 (DOCK7-Ab), or an anti-p116Rip (p116Rip-Ab) antibody and then were analyzed by immunoblotting with antibodies to DOCK7 and p116Rip. Molecular masses are shown in kilodaltons. (G and H) Colocalization of DOCK7 and p116Rip in V-SVZ–derived neuroblasts. (G) Coronal sections of P10 mouse forebrain at position III in the RMS as indicated in the cartoon in Fig. 1 A immunostained with antibodies to DOCK7 and p116Rip and counterstained with DAPI. Boxed regions are enlarged and shown at the bottom. (H) Neuroblasts dissociated from V-SVZ tissue of P1–3 mice were fixed at 30 h in vitro, followed by immunostaining with indicated antibodies and counterstained with DAPI. Bars: (C and G, top) 50 µm; (G, bottom, and H) 10 µm.

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