Figure 1

Validation of C/EBPδ as a target of p63. A. Evaluation of C/EBPα, C/EBPβ and C/EBPδ, by semi-quantitative RT-PCR analysis in control and cells treated with siRNA of p63. β-actin was used as an internal control. In the lower Panel, primary human keratinocytes (KCs) were treated similarly and RT-PCR performed for ΔNp63α and C/EBPδ; the invariant GAPDH was included as internal control. B. Confocal microscopy immunofluorescence analysis of primary KCs treated with control and p63 RNAi oligos. Fixation and staining with the indicated antibodies was performed at 48 hours post-transfections. Note that the laser settings of the C/EBPδ image of p63-inactivated cells had to be lowered considerably due to extemely strong fluorescence. C. Chromatin Immunoprecipitation analysis of the C/EBPδ-1 Kb (Indicated by the square box), using HaCaT cells (Left Panel) and the indicated antibodies: NF-YB, p63 (Santa Cruz H137; Dako 4A4) and control -Ctl- anti-Flag antibodies (Sigma). Right Panel, ChIPs with primary KCs, anti-p63 4A4 (Dako), anti-p63α specific polyclonal [19], anti-p53 (Ab7 Calbiochem). As a control, ChIPs from KCs were used to amplify the α-globin promoter. In the bottom part, the p53 and p63 sites at -1000 of the human C/EBPδ promoter are shown, in an area of conservation with the mouse gene. HBA1 is the human α-globin promoter.