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Fig. 4. IKK regulation by GATA-3, and overexpression in Np63/TAp63 genetically complemented mice. (A) Galactosidase assay showing the ability of TAp63 and Np63 to activate the GATA-3 promoter. The ratio of GATA-3 promoter to transcription factor is 1:3. The in vitro transcription assay shown was performed in Saos-2 cells, and similar results were also obtained in HEK293 cells (data not shown). Results are shown as mean ± s.d. from three independent experiments. (B) ChIP showing the binding of p53 and p63 proteins to the GATA-3 promoter. Lanes 1 and 2, input; lanes 3 and 4, specific antibody (Sp-IP, anti-GATA-3 and anti p21); lanes 5 and 6, non-specific antibody (Not Sp-IP). A representative result of two independent experiments is shown. (C) Luciferase assay showing the ability of GATA-3 to directly induce the IKK promoter. The ratio of IKK promoter to transcription factor is 1:1 and 1:3. In lanes 8 and 9 the ratio was 1:1:1. The in vitro transcription assay shown was performed in HaCaT cells, and similar results were also obtained in HEK293 cells (data not shown). Results are shown as mean ± s.d. from three independent experiments. (D) Western blot of epidermal cell extracts from p63^–/–, p63^–/–;TA, p63^–/–;N, p63^–/–;N;TA mice (see 13). Double p63^–/–;N;TA complemented mice, as well as p63^–/–;TA mice, show higher levels of IKK expression in vivo compared with p63^–/– mice. As loading control we detected actin. Lower panel shows the normalisation of the induction of IKK to actin protein level.

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