Fig. 7. Jag1 is a direct target gene of ß-catenin. (A-E) Whole mounts of K14N ß-cateninER tail epidermis labelled with antibodies to K14 (red) and jagged 1 (green). Mice were untreated (A) or treated for 21 days with 4OHT and either examined immediately (B) or after 21 days without 4OHT treatment (C). Following 21 days without treatment, mice received 4OHT for 7 days (D) or 14 days (E). (F,G) Immunostaining for jagged 1 (red) in back skin sections from K14N ß-cateninER mice treated with 4OHT (F) or 4OHT + DAPT (G). Scale bars: 100 µm (A-G). (H-J,L,M) Immunostaining for jagged 1 (green) in back skin sections (H-J) and tail epidermal whole mounts (L,M) of wild-type (H,I,L) and K14NLef1 (J,M) mice. H is a control with no primary antibody. DAPI (blue) is shown at lower magnification (F-J). Dashed lines in L,M indicate hair follicles and sebaceous glands. Scale bars: 100 µm in H-J,L,M. (K) Wild-type or K14N ß-cateninER mouse keratinocytes were cultured with or without 4OHT for 72 hours, then co-transfected with pRL (Renilla luciferase) and different ratios of empty vector (ev), mNotch1^ICD (NICD) and Hes1 luciferase (Hes1-Luc). Data are expressed as average light units ±s.d., relative to the renilla luciferase control. ^*P<0.01. n, number of replicate samples. (N) Location of putative Tcf/Lef-binding sites in the mouse Jag1 promoter. (O) RT-PCR of Jag1 and ß-actin transcripts from wild-type and K14N ß-cateninER mouse keratinocytes treated with acetone or 4OHT in the presence of cycloheximide. Three independent experiments were analysed with consistent replicates and consistent results among experiments. (P) Wild-type and K14N ß-cateninER mouse keratinocytes were fixed and subjected to chromatin immunoprecipitation using anti-ß-catenin or control antibodies. Primers surrounding the putative Tcf/Lef-binding sites or an irrelevant region of the Jag1 promoter (831-370) were used for the PCR analysis.