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Figure 3


Fig. 3. The Delta1 PDZ domain binds syntenin and promotes keratinocyte cohesiveness. (A) GFP-labelled clones formed by keratinocytes expressing the Delta constructs shown 5 days after seeding within a confluent sheet of unlabelled WT keratinocytes. Bar, 20 µm. (B) Percentage of cohesive colonies. Data are mean ± s.d. of five experiments. Values significantly different from WT control are marked with asterisks (Student's t-test, ***P<0.001 or **P<0.05). (C) Western blots of A431 cells transfected with empty vector (ev) or the Delta constructs shown following pull down with GST (control; right panel) or GST-syntenin (left panel). Blots were probed with anti-ZfDelta or anti-GST antibodies. (D) Western blot of untransfected A431 cell lysates following pull down with GST or GST-syntenin (GST-syn). Blot was probed with antibody to human Delta1. Total cell lysates (Lys) served as a positive control. (E) A431 cell lysates immunoprecipitated with antibodies against syntenin ({alpha}-syn) or laminin5 ({alpha}-Lam) followed by western blotting with anti-Delta1 antibody. Lys, lysate subjected to western blotting without prior immunoprecipitation. (F) Immunofluorescence staining of human primary keratinocytes with anti-syntenin antibody (green). DAPI was used as a nuclear counterstain (blue). Bar, 10 µm. (G) Immunofluorescence staining of human interfollicular epidermis with an anti syntenin antibody. Horizontal lines mark position of putative stem cell clusters. Bar, 50 µm.





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