Fig. 2. Effects of Shh deficiency or SMO inhibition on GLI protein expression. (A-L) Immunohistochemistry of 4 µm tissue sections generated from E14.5 kidney tissue using specific anti-GLI antibodies. The substrate reaction generated a red color. Tissue was counterstained with Hematoxylin (blue). In tissue generated from wild-type mice (A,E,I), GLI1, GLI2 and GLI3 were each detected in metanephric-derived epithelial structures and ureteric bud branches. Shh deficiency decreased GLI1 and GLI2 expression compared with wild type (D,H versus A,E). By contrast, total GLI3 expression in kidney was not decreased in Shh^–/– mice (L). Antibody specificity was demonstrated by lack of staining in corresponding Gli-deficient mice (B,C,F,G,J,K). Scale bar: 100 µm. (M) Western analysis of E14.5 kidney tissue lysates generated from wild-type and Shh^–/– mice. Shh deficiency decreased GLI1 and GLI2. Although GLI3 activator (190 kDa) was decreased, GLI3 repressor (89 kDa) was unaffected compared with wild type. The ratio of GLI3 activator to GLI3 repressor in renal tissue was markedly decreased from 3.25 in wild type to 0.23 in Shh^–/– mice. (N) Western analysis of tissue lysates generated from cultured embryonic kidneys isolated from E11.5 wild-type mice and treated for 4 days with culture medium alone or with culture medium supplemented with drug vehicle (100% ethanol), SHH-N or cyclopamine. In the presence of culture medium, embryonic kidneys expressed GLI1, GLI2 and GLI3 activator (190 kDa) in excess of GLI3 repressor (89 kDa) (GLI3 activator: GLI3 repressor=3.68). Treatment with SHN-N increased GLI1, GLI2 and the relative expression of GLI3 activator versus GLI3 repressor (GLI3 activator:GLI3 repressor=11.9). Treatment with drug vehicle had no significant effect on GLI protein expression. By contrast, cyclopamine decreased GLI1 and GLI2 and the relative expression of GLI3 activator versus GLI3 repressor (GLI3 activator:GLI3 repressor=1.43).