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. 2010 Nov 29;191(5):1029–1041. doi: 10.1083/jcb.201007008

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© 2010 Biswas et al.

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Figure 4. — Pcdh19 physically interacts with Ncad. (A) Diagram of the constructs used for coIPs and BiFC. We constructed plasmids containing full-length Pcdh19, Pcdh19 lacking the cytoplasmic domain (Pcdh19ΔCP) or the cytoplasmic domain of Pcdh19 fused to human CD4 (CD4-Pcdh19CP), and the corresponding Ncad plasmids (Ncad, NcadΔCP, and CD4-NcadCP). (B–D) CoIPs show that Pcdh19 interacts with Ncad when cotransfected into COS cells. (B) Immunoprecipitation of Pcdh19-myc pulls down Ncad-GFP. (C) Immunoprecipitation of Ncad-GFP brings down Pcdh19-myc. (D) Immunoprecipitation of Ncad-myc also pulls down Pcdh19-GFP. (E) The cytoplasmic domains of Ncad and Pcdh19 are not required for their interaction. Immunoprecipitation of Pcdh19ΔCP-myc pulled down NcadΔCP-GFP. (F and G) The extracellular domains of Pcdh19 and Ncad are required for their interaction, as immunoprecipitation of CD4-NcadCP-myc fails to pull down CD4-Pcdh19CP-GFP (F), and pulling down CD4-Pcdh19CP-GFP does not coimmunoprecipitate CD4-NcadCP-myc (G). (H) Immunoprecipitation of endogenous Pcdh19 pulls down classical cadherins in vivo. Zem2S extracts and embryo extracts were immunoprecipitated with (+) or without (−) anti-Pcdh19 primary antibody, and proteins were blotted with anti–pan-cadherin antibody. (I–L) Bimolecular fluorescence complementation performed in HEK293 cells. Green, Venus fluorescence; blue, DAPI. (I and J) Pcdh19 and Ncad directly associate in cells transfected with the full-length constructs Pcdh19-VenusC and Ncad-VenusN (I) or with NcadΔCP-VenusN and Pcdh19ΔCP-VenusC (J). (K and L) HEK293 cells transfected with Ncad-VenusN and CD4-Pcdh19-VenusC (K) or CD4-NcadCP-VenusN and CD4-Pcdh19CP-VenusC (L) do not exhibit any fluorescence. (M and N) A maximum intensity projection of two optical sections of live cells expressing Pcdh19-GFP (M) or Ncad-GFP (N) in a five-somite stage embryo. (O) Low power image of two five-somite stage embryos coexpressing Ncad-VenusN and Pcdh19-VenusC. Fluorescence is evidence of efficient and widespread interaction of Pcdh19 and Ncad. (P) A maximum intensity projection of two optical sections of cells coexpressing Ncad-VenusN and Pcdh19-VenusC. Arrows point to accumulations at cell–cell contacts. Bars: (I and J) 25 µm; (K and L) 100 µm; (O) 150 µm; (M, N, and P) 10 µm.