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Fig. 6. GLI3 controls GLI1 and GLI2 expression via transcriptional
mechanisms. (A) Western analysis of E14.5 kidney tissue lysates
using specific anti-GLI antibodies. In Shh–/–
mice, expression of GLI1 and GLI2 was decreased compared with wild type and
Gli3–/–. In
Shh–/–;Gli3–/– mice,
GLI1 and GLI2 expression was rescued to wild-type levels. (B) Agarose
gel electrophoresis of products generated by RT-PCR using RNA isolated from
E14.5 embryonic kidney. Expression of Gli1 and Gli2 mRNA was
decreased in Shh–/– compared with wild type
and Gli3–/– and was rescued in
Shh–/–;Gli3–/– mice.
(C) Identification of GLI consensus binding sequences in the 5'
flanking region of mouse Gli1 and Gli2. Nucleotides
represented in upper case are exact matches to those identified previously in
GLI consensus binding sequences (see text). Arrows indicate promoter segments
amplified by PCR during chromatin immunoprecipitation (see below). (D)
PCR products identified by agarose electrophoresis after chromatin
immunoprecipitation using E11.5 wild-type kidney tissue cultured for 4 days in
the presence of drug vehicle or cyclopamine. In vehicle-treated embryonic
kidneys, GLI1 and/or GLI2 bound 5' flanking regions containing GLI
consensus binding regions in Gli1 and Gli2. No binding of
GLI3 with this region in Gli1 was detectable. Cyclopamine decreased
GLI1 and GLI2 binding and induced binding of GLI3 to each promoter region.
Neg, DNA amplified after immunoprecipitation with non-immune serum; Pos, DNA
amplified using genomic DNA and specific primers; Input, DNA amplified from
cross-linked DNA before immunoprecipitation.
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