Journal: Nature communications

Article Title: Speed control for neuronal migration in the postnatal brain by Gmip-mediated local inactivation of RhoA.

doi: 10.1038/ncomms5532

Figure Lengend Snippet: Figure 1 | Interaction of Gmip with Girdin in postnatal mouse new neurons. (a) Expression of Gmip in the postnatal mouse brain. Lysate of V-SVZ tissue from P5 mice was subjected to immunoblotting using an anti-Gmip antibody. The Gmip protein is indicated by an arrowhead. (b,c) Complex formation between endogenous Gmip and Girdin in brain lysate. The lysate of V-SVZ tissue from P2 rats was subjected to immunoprecipitation (IP) analysis with control IgG or an anti-Girdin antibody, followed by western blot (WB) analysis with anti-Gmip antibody (b). Reciprocally, the same lysate was subjected to IP with control IgG or an anti-Gmip antibody, followed by WB analysis with anti-Girdin antibody (c). Immunoprecipitated proteins are indicated by arrowheads. (d–j) Expression pattern of Gmip in the postnatal V-SVZ, RMS and OB. Coronal sections from P7 mouse brain were stained with anti-Gmip antibody (red) in combination with the indicated antibodies (green). Arrows, Gmip+NeuN- cells (j). (k) Localization of Gmip and Girdin in cultured new neurons from the V-SVZ. New neurons dissociated from the V-SVZ tissue of Girdin / or WT littermate (Girdinþ / þ) P1–4 mice were fixed at 2 days in vitro, followed by immunostaining with the indicated antibodies (Gmip: red, Girdin: green and bIII-tubulin: cyan). Arrows, proximal leading process. Scale bars, 20 mm (d–j) and 10 mm (k). For full blots, see Supplementary Fig. 10.

Article Snippet: Other antibodies used in this study included sheep anti-Girdin polyclonal antibody (1:100, R&D Systems, Minneapolis, MN), rabbit anti-EGFR polyclonal antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-cleaved caspase-3 antibody (1:200, Cell Signaling Technology, Beverly, MA), goat anti-Dcx polyclonal antibody (1:100, Santa Cruz Biotechnology), rabbit anti-Dcx polyclonal antibody (1:200, Cell Signaling Technology), mouse bIII-tubulin antibody (1:1,000, Sigma, St Louis, MO), rabbit anti-GFP polyclonal antibody (1:1,000, MBL, Nagoya, Japan), rat anti-GFP polyclonal antibody (1:200, Nacalai tesque, Kyoto, Japan), rabbit anti-DsRed antibody (1:1,000, Clontech Laboratories, Inc., Mountain View, CA), mouse anti-b-actin monoclonal antibody (1:500, Roche, Basel, Switzerland), sheep anti-BrdU antibody (1:1,000, Fitzgerald Industries International, Acton, MA), mouse anti-NeuN monoclonal antibody (1:100, Millipore, Billerica, MA), normal sheep IgG, and normal rabbit IgG (10 mg ml 1, Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Immunoprecipitation, Control, Staining, Cell Culture, In Vitro, Immunostaining