Figure 4.

Cld-12 in brain blood vessels. (a) Specificity of newly generated anti–Cld-12 pAb. Immunoblotting of total lysates of Escherichia coli expressing GST fusion proteins with cytoplasmic domains of Cld-1–16 confirmed its specificity. CBB, Coomassie brilliant blue staining; anti–Cld-12 pAb, immunoblotting with anti–Cld-12 pAb. (b) Frozen sections of the wild-type and Cld5 ^−/− brains of 18.5-d embryo were double stained with anti–ZO-1 mAb and anti–Cld-5 pAb or anti–Cld-12 pAb. Cld-5 was completely undetectable from the Cld5 ^−/− brain. Cld-12 was concentrated at ZO-1–positive TJs not only in the wild-type but also in the Cld5 ^−/− brain endothelial cells. These Cld-12 signals were abolished when the anti–Cld-12 pAb was preincubated with the GST-Cld-12 fusion protein. There appeared to be no significant difference in the intensity of Cld-12 signal between the wild-type and Cld5 ^−/− brain endothelial cells. The concentration of occludin at TJs of endothelial cells was not affected in the Cld5 ^−/− brain (unpublished data). Bar, 20 μm.