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Fig. 8. Inability of Necl-1 to bind afadin. (A) Yeast two-hybrid assay. 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. (B) Coimmunoprecipitation assay. FLAG-tagged Necl-1 was immnunoprecipitated with anti-FLAG mAb from the extract of Necl-1-L cells. For the control experiments, nectin-2 was immunoprecipitated with anti-nectin-2 mAb from the extract of nectin-2-L cells. Immunoprecipitates were then subjected to SDS-PAGE (10% polyacrylamide gel), followed by western blotting with anti-FLAG, anti-nectin-2 or anti-afadin mAbs. (C) Affinity chromatography. GST-Necl-1-CP or GST-nectin-3-CP (2 nmol of protein each) was applied to amylose resin beads on which MBP-afadin-PDZ (200 pmol of protein each) was immobilized. After the beads were extensively washed, the bound proteins were subjected to SDS-PAGE (13%), followed by protein staining with Coomassie brilliant blue. The results shown are representative of three independent experiments.
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