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. 1999 May 3;145(3):539–549. doi: 10.1083/jcb.145.3.539

Figure 2.

Figure 2

Direct binding of l-afadin to nectin in vitro. (A) Affinity chromatography. GST-l-afadin-PDZ (aa 1007–1125) or GST alone (20 μg of protein each) was applied to protein A–Sepharose beads on which Myc-nectin-1-CP (aa 379–518) was immobilized through the anti-Myc mAb. After the beads were extensively washed, the bound proteins were subjected to SDS-PAGE (12% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue. Arrow, GST-l-afadin-PDZ; arrowhead, Myc-nectin-1-CP. (B) 35S-Labeled l-afadin blot overlay. GST-fusion proteins of the cytoplasmic regions of nectin (0.3 μg of protein each) were subjected to SDS-PAGE (12% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue or by 35S-labeled l-afadin blot overlay. (B1) Protein staining. (B2) 35S-Labeled l-afadin blot overlay. GST-Nectin-1-CPC, aa 449–518; GST-Nectin-1-CPC-ΔC, aa 449–514; GST-Nectin-2α-CP, aa 387–467; GST-Nectin-2α-CP-ΔC, aa 387–463; GST-Nectin-2δ-CP, aa 403–530; GST-Nectin-2δ-CP-ΔC, aa 403–526.