Fig. 6.

Crim1 regulates integrin and FAK and ERK phosphorylation in lens development. (A) Colocalization of β1 integrin and Crim1 (arrows) at LE cell-cell adhesions and at the basal surface of LE cells. (B) Co-immunoprecipitation of β1 integrin with His-Myc-tagged Crim1-FL in HEK 293T cells. (C,D) Immunostaining of active (9EG7) β1 integrin shows decreased staining in Crim1^glcr11 (C) and Crim1^null (D) lenses. Arrows point to the LE cell basement membrane. Phalloidin stains the actin cytoskeleton; DAPI stains nuclei. Quantification of fluorescence intensity is shown beneath. n=3. (E) Western blot analysis of P6 wild-type and Crim1^glcr11 lenses (left) and E18.5 control and Crim1^null lenses (right) with the indicated antibodies. n=5. (F) 21EM15 cells were treated with either of two siRNAs directed against Crim1 for 48 h and then cell lysates were western blotted with the indicated antibodies. Each bar represents the mean of triplicates. (G) Compound heterozygous P0-3.9-GFPCre;Crim1^flox/+;Itgb1^flox/+ mice exhibit iris coloboma (arrows) and abnormal lens morphology at E18.5, and later develop bilateral cataract at P21 (arrowheads). n=6; all six P21 compound heterozygotes obtained were affected, as compared with none of four littermate controls. Arrowhead in P21 section indicates LE cell detachment from LF cells. Red, Phalloidin-stained actin cytoskeleton. Blue, DAPI-stained nuclei. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). Scale bars: 10 μm.