detection kit (Roche). Sections from the middle of the wound were also stained using the Masson trichrome procedure (Sigma-Aldrich). Stained tissues were analyzed on a confocal scanning laser microscope (LSM; Carl Zeiss MicroImaging, Inc.). In situ hybridization of parafﬁn sections was performed using digoxygenin-labeled RNA probes (Roche; Huelsken et al., 2001). The anti- sense transcripts of mouse cDNAs were as follows: a 1.4-kb HGF/SF frag- ment that encompasses the 3′ coding sequence, a 0.7-kb HGF/SF fragment that encompasses the 5′ coding sequence, and a 3.7-kb c-Met fragment. Western blot analysis was performed using antibodies speciﬁc to Erk1/2, phospho Erk1/2, Akt, phospho-Akt, phospho-Gab1, and phospho-PAK1/2 (Cell Signaling Technology). Laser capture microdissection and Southern blotting For laser capture microdissection, 46 wound areas were excised, embed- ded in Tissue-Tek OCT compound and snap-frozen in liquid nitrogen. Fro- zen sections (8 μm) were prepared and ﬁxed in 70% EtOH for 60 s and stained shortly with hematoxylin/eosin. Laser capture microdissection was performed using an Arcturus PixCell II apparatus. Southern blot analysis of hyperproliferative epithelia from 400 microdissected sections was per- formed according to standard procedures. Primary keratinocyte culture, in vitro scratch assay, and immunoﬂuorescence Primary murine keratinocytes were isolated from newborn mice, as de- scribed previously (Caldelari et al., 2000), and cultured on collagen IV– coated plates in deﬁned keratinocyte serum-free medium (Invitrogen). The conﬂuent monolayers of primary keratinocytes were scratch wounded by a pipette tip and further cultured in the presence of growth factors: 10 U/ml HGF/SF (Weidner et al., 1993) and 20 ng/ml TGFα (Sigma- Aldrich). For immunostaining, cells were washed with PBS and ﬁxed in 4% formaldehyde in PBS for 15 min at room temperature. The following anti- bodies were used: antivinculin (Sigma-Aldrich); antipaxillin (BD Biosciences); anti–phospho-c-Met, anti-RhoA, anti–γ-tubulin (Santa Cruz Biotechnology); and anti-VASP (Cell Signaling Technology). Additional reagents used were TRITC/FITC phalloidin (Sigma-Aldrich) and YO-PRO (Invitrogen). Online supplemental material Fig. S1 shows the expression of c-Met in bulge stem cells. Fig. S2 shows isolated keratinocytes from control and conditional c-Met mutant mice. The online version of this article is available at http://www.jcb.org/ cgi/content/full/jcb.200701086/DC1. We thank Ute Schaeper (Berlin) and Daniel Besser (Berlin) for their helpful ad- vice and discussions in the course of the work, and Monika Krampert (Zurich) and Klemens Rottner (Braunschweig) for valuable experimental advice and discussions. We also thank Johannes Fritzmann (Berlin) for his help with the laser capture microdissection experiment. 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