Fig. 1. Construction, expression and activity of the K14NLef1 transgene. (A,D) Schematic representations of (A) the K14NLef1 transgene construct and (D) the structure of a hair follicle. (B) Immunoblot of epidermis from wild-type (WT) and transgenic (TG) mice using anti-Myc-tag to detect the transgenic protein and anti-actin as a loading control. (C) Semi-quantitative RT-PCR for endogenous Lef1 (WTs + as), transgenic NLef1 (TGs + as) and HPRT of RNA isolated from primary wild-type (wt) or transgenic (tg) keratinocytes. Left-hand lane in each gel shows molecular weight markers and lanes labelled H show controls in which no RNA was added. PCR reactions were analysed after (left to right) 10, 25, 35 or 65 (65 not shown for HPRT) rounds of amplification. (E) Luciferase reporter assay. Keratinocytes were isolated from wild-type (WT) and transgenic (TG) animals and transfected with pTOPFLASH (TOP) or pFOPFLASH (FOP). In some cases, cells were co-transfected with the (ßcat/Lef1 fusion construct (Fusion), N-terminally truncated ß-catenin (T2) or Tcf-3 (TCF). Luciferase activity values are shown in light units and represent the average of triplicate determinations, corrected for transfection efficiency. (F) Immunoblot of total and Triton-soluble protein lysates of primary keratinocytes isolated from wild-type (wt) and K14NLef1 (tg) mice with antibodies to E-cadherin, ß-catenin, plakoglobin and actin (loading control). (G) Flow cytometry of primary keratinocytes from wild-type (wt) and K14NLef1 (tg) mice labelled with anti-ß1 integrin antibody or secondary antibody alone.

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