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Fig. 1. Expression of N17Rac1 in the epidermis of mice and in primary murine keratinocytes. (a) Schematic representation of the transgenic construct. (b,c) PCR (b) and Southern blot (c) analysis of tail snips from transgenic (tg) and wild-type (wt) mice. Amplified or excised plasmid fragments were used as positive controls (+). (d,e) Immunostaining of skin samples (d) and western blot analysis of adult primary keratinocytes (e) from tg and wt mice with the anti-Myc antibody 9B11. Note expression of N17Rac1 in basal epidermal and outer root sheath keratinocytes of tg mice (green). Bar, 100 µm. (f) Western blot analysis of protein extracts from cultured tg and wt keratinocytes with a monoclonal antibody against Rac (upper panel). The mutant Rac protein (N17Rac1) is Myc-tagged and, therefore, migrates slower compared with endogenous Rac. Loading controls show actin expression (lower panel). (g) Western blot analysis with the anti-Myc antibody 9B11 of protein extracts from cultured wt and tg keratinocytes with strong (se) and weak (we) expression of the transgene, respectively (upper panel). Loading controls show actin expression (lower panel). (h,i) Results of CFE assays with wt (n=4), strong tg (se; n=4) and weak tg (we; n=3) keratinocytes. Bar graphs show number of cells per colony (h) and colony area (µm²x10⁴) (i); error bars show standard deviation (± s.d.). ^*P<0.05; ^**P<0.01; ^***P<0.001; ns, not significant.

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