Figure 7.

Generation of afadin^−/− ES cells. (A) Restriction maps of the afadin^+/− allele, the second targeting vector, and the afadin^−/− allele of the afadin gene. Filled boxes, exons. S, SacI; H, HindIII; Sp, SphI; and X, XbaI. (B) Southern blot analysis. HindIII-digested DNAs derived from ES cells were hybridized with the 5′ or 3′ probe. (C) Western blot analysis. A cell lysate of ES cells, simple EBs, or cystic EBs (20 μg of protein each) was subjected to SDS-PAGE (8% polyacrylamide gel), followed by Western blot analysis using the anti–l-afadin mAb or the mAb recognizing both l- and s-afadins. sEBs, simple EBs. cEBs, cystic EBs. Black arrowheads indicate l-afadin and white arrowhead indicates s-afadin.