Fig. 4. Reduced BrdU incorporation and ERK phosphorylation in Rac1^E-KO and N17Rac1 transgenic (tg) keratinocytes in vivo and in vitro. (a-c) Numbers of BrdU-positive keratinocytes in sections of wounded skin (a,c) 5 days or (b) 2 days after wounding as the average number of BrdU-positive keratinocytes per microscopic field ± s.d. representative for (a) five or (b) seven female wild type (wt) mice, (a) six or (b) eight female N17Rac1 tg mice and (c) eight Rac1^E-HET versus 11 Rac1^E-KO mice (mixed sex groups). (d) Percentage of BrdU-positive nuclei in keratinocyte cultures obtained from wt and N17Rac1 tg mice in serum-free low-Ca²⁺ FAD medium supplemented with 10 ng/ml EGF (dark gray bars) or 100 ng/ml IGF1 (light gray bars) showing the average number of BrdU-positive nuclei ± s.d. (representative for six different N17Rac1 tg and five wt cell lines; n=6). (e) Top: Micrographs showing immunostaining of sections of skin wounds from Rac1^E-KO mice (Rac1^E-KO) and wt mice using an antibody against phosphorylated ERK1/2. Yellow lines indicate the wound margin; arrows point towards the tips of the tongues; dashed lines indicate basement membrane. Bar, 40 µm. Bottom: Western blot analysis of phosphorylated ERK (pERK) in N17Rac1 tg and wt keratinocytes plated on fibronectin (fn) and collagen I (col I), respectively. Loading controls show actin expression. (f) Western blot analysis of phosphorylated ERK (pERK) in N17Rac1 tg and wt keratinocytes unstimulated (–) or stimulated (+) with 10 ng/ml EGF. Total ERK (ERK) is shown as loading control. The densitiy of the phosphorylated band relative to the loading control is shown in the lower panel (in %). ^**P<0.01; ^***P<0.001; n=3, six different transgenic cell lines.