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Fig. 9. Binding of Dlg3, Pals2 and CASK to Necl-1. (A) Yeast two-hybrid analysis of the binding of Dlg3, Pals2 and CASK to Necl-1 and Necl-2. Yeast transformants with the indicated plasmids were streaked on synthetic complete medium lacking adenine to score the ADE2-reporter activity and incubated at 30°C for 3 days. pGAD10-Dlg3 (lacking the N-terminal 1-5 aa) was a positive clone which was isolated by yeast two-hybrid screening of pGBD-C1-Necl-1-EC from a rat brain library. (B) Coimmunoprecipitation of Dlg3, Pals2 or CASK with Necl-1 or Necl-2. HEK293 cells were co-transfected with the indicated plasmids and FLAG-tagged Necl-1, Necl-1-C, Necl-2 or Necl-2-C were immunoprecipitated with anti-FLAG mAb from the extract of co-transfected HEK293 cells, respectively. The immunoprecipitates were then subjected to SDS-PAGE (10%), followed by western blotting with anti-FLAG, anti-HA or anti-Myc mAbs. (C) Coimmunoprecipitation of Necl-1 with Dlg3 from mouse cerebellum. Dlg3 was immunoprecipitated with anti-Dlg3 antiserum from an extract of mouse cerebellum. Non-immune rabbit serum was used for the control experiments. The immunoprecipitates were subjected to SDS-PAGE (10%), followed by western blotting with affinity-purified anti-Dlg3 or anti-Necl-1 pAbs. (D) Necl-1-dependent recruitment of Dlg3, Pals2 or CASK to cell-cell contact sites. Necl-1-L (a,c,e) or Necl-1-C-L cells (b,d,f) were transfected with pCMV-HA-Dlg3 (a,b), pCMV-HA-Pals2 (c,d) or pEFBOS-Myc-CASK (e,f), and double-stained with anti-FLAG mAb (green, a1-f1) and anti-HA mAb (red, a2-d2), or anti-Myc pAb (red, e2,f2). Arrows indicate cell-cell contact sites. Bars, 10 µm. The results shown are representative of three independent experiments.

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