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Fig. 5. Reduced adhesion and spreading of N17Rac1 transgenic (tg) keratinocytes on collagen I in vitro. (a) Rac1-GTP loading in keratinocytes 40 minutes after plating onto collagen I (col I)- or fibronectin (fn)-coated dishes. Active Rac (Rac1_GTP) was precipitated using biotin-CRIB peptide and analysed by western blotting with anti-Rac antibody (upper panel). Total (active and inactive) Rac1 protein (Rac1_total) is shown in the lower panels as a loading control (n=4). Determination of signal intensity for Rac1-GTP relative to Rac1_total using densitometry revealed no consistent difference between the levels of Rac1-GTP in keratinocytes plated onto collagen I compared with fibronectin. (b) Percentage of adherent tg and wild type (wt) keratinocytes on collagen I (gray bars) and fibronectin (white bars) 1 hour after plating. Error bars give the mean ± s.d. of four different tg and wt keratinocyte lines each (n=4). (c) Micrographs showing immunostaining of Myc and polymerized actin in wt and tg keratinocytes 40 minutes after plating onto collagen-I-coated dishes. (d,e) Histograms showing cell spreading of four different N17Rac1 tg (red) and three different wt (black) keratinocyte lines on (d) collagen I and (e) fibronectin. Cell size is given in arbitrary units (n=3). ^**P<0,01; ns, not significant.

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