30 V in Tris/Glycine buffer with 20% methanol and transfer was confirmed with Ponceau Red (Sigma) staining of the membranes. After de-staining in distilled water, membranes were incubated in blocking buffer (5% powdered milk in TBS) for 30 minutes at room temperature. Membranes were then incubated with 1u antibody overnight at 4uC. After rinsing in TBS containing 0.1% Tween-20 (365 minutes), membranes were incubated with 2u antibody for 1 h at room temperature, rinsed in TBS with 0.1% Tween-20 (365 minutes), and developed (ECL Chemilumines- cence; GE Healthcare). Antibodies used were as follows; anti- phospho-p44/42 MAPK (Cat. #4370, Cell Signaling, New England Biolabs, Hitchin, UK) and anti-MAPK1 (Cat. #05- 957, Upstate, Millipore). Primary antibodies were diluted 1:1000 in TBS +3% BSA and incubate overnight at 4uC. Secondary antibodies (Dako), coupled with horseradish peroxidase, were used at a 1:1000 dilution, incubated for 1 h at room temperature. Densitometric analysis was performed using ImageJ 1.429 software. Signal density was normalized to anti-ERK antibody as a loading control, for at least three separate treatments. RNA Interference SiRNA used (Dharmacon, Cramlington, UK): Genome smart pool for human FGF22, M-013171. Cells (40–50% confluent in 6- well plates) were transfected for 4 h with siRNA (10 nM) using 4 ml Interferin (Polyplus transfection; PeqLab, Fareham UK), in a total reaction volume of 1.1 ml in each well of a 6-well plate. Messenger RNAs and functional activity were assayed 48 h post- transfection, and compared with mock and/or control siRNA- treated cells (SiControl Non-Targeting siRNA pool; D-001210 Dharmacon). References 1. Ornitz DM, Itoh N (2001) Fibroblast growth factors. Genome Biol 2: REVIEWS3005. 2. Itoh N, Ornitz DM (2010) Fibroblast growth factors: from molecular evolution to roles in development, metabolism and disease. J Biochem. 3. Ornitz DM, Xu JS, Colvin JS, McEwen DG, MacArthur CA, et al. (1996) Receptor specificity of the fibroblast growth-factor family. J Biol Chem 271: 15292–15297. 4. Zhang X, Ibrahimi OA, Olsen SK, Umemori H, Mohammadi M, et al. (2006) Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family. J Biol Chem 281: 15694–15700. 5. Igarashi M, Finch P, Aaronson S (1998) Characterization of recombinant human fibroblast growth factor (FGF)-10 reveals functional similarities with keratinocyte growth factor (FGF-7). J Biol Chem 273: 13230–13235. 6. Min H, Danilenko DM, Scully SA, Bolon B, Ring BD, et al. (1998) Fgf-10 is required for both limb and lung development and exhibits striking functional similarity to Drosophila branchless. Genes Dev 12: 3156–3161. 7. Sekine K, Ohuchi H, Fujiwara M, Yamasaki M, Yoshizawa T, et al. (1999) Fgf10 is essential for limb and lung formation. Nat Genet 21: 138–141. 8. De Moerlooze L, Spencer-Dene B, Revest J, Hajihosseini M, Rosewell I, et al. (2000) An important role for the IIIb isoform of fibroblast growth factor receptor 2 (FGFR2) in mesenchymal-epithelial signalling during mouse organogenesis. Development 127: 483–492. 9. Guo L, Degenstein L, Fuchs E (1996) Keratinocyte growth factor is required for hair development but not for wound healing. Genes Dev 10: 165–175. 10. Zhang H, Dessimoz J, Beyer TA, Krampert M, Williams LT, et al. (2004) Fibroblast growth factor receptor 1-IIIb is dispensable for skin morphogenesis and wound healing. Eur J Cell Biol 83: 3–11. 11. Nakatake Y, Hoshikawa M, Asaki T, Kassai Y, Itoh N (2001) Identification of a novel fibroblast growth factor, FGF-22, preferentially expressed in the inner root sheath of the hair follicle. Biochim Biophys Acta 1517: 460–463. 12. Beyer TA, Werner S, Dickson C, Grose R (2003) Fibroblast growth factor 22 and its potential role during skin development and repair. Exp Cell Res 287: 228–236. 13. Schlake T (2005) FGF signals specifically regulate the structure of hair shaft medulla via IGF-binding protein 5. Development 132: 2981–2990. 14. Hamada K, Ozawa K, Itami S, Yoshikawa K (1999) Human fibroblast growth factor 10 expression in dermal papilla cells, outer root sheath cells and keratinocytes. Exp Dermatol 8: 347–349. FGF22 in Skin Development and Repair Supporting Information Figure S1 RNAi-mediated knock down of FGF22 in HaCaT cells. HaCaT cells were subject to mock transfection (Ctr) or transfection with 10 nM control non-targeting siRNA (Scr) or siRNA to FGF22 (1 nM or 10 nM). Messenger RNA levels for FGF22 and NRF2 were measured by Realtime PCR, normalised to GAPDH. Samples were run in triplicate and a representitive repeat is shown. FGF22 RNA levels were reduced by 50% following RNA interference, but levels of NRF2 expression were unaffected. (TIF) Figure S2 Expression of Fgf7 and Fgf10 mRNA in fgf22 wild type and knockout mice. 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(2010) Fibroblast growth factor receptors 1 and 2 in keratinocytes control the epidermal barrier and cutaneous homeostasis. J Cell Biol 188: 935–952. 26. Sundberg JP, Hogan ME (1994) Hair Types and Subtypes in the Laboratory Mouse. In: Sundberg JP, editor. Handbook of Mouse Mutations with Skin and Hair Abnormalities: Animal Models and Biomedical Tools. Bar Harbor, Michigan: CRC Press. 57–68. PLoS ONE | www.plosone.org 11 June 2012 | Volume 7 | Issue 6 | e39436