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Files in this Data Supplement:
Fig. S1. Hematoxylin and Eosin-stained sections through the skin of embryos 14 days post virus injection (HH41). Embryos were infected with a control virus (A), RCAS-R1 (B), or RCAS-R2 (C). HE staining of 6 mm paraffin sections reveals a thinner epidermis in both RCAS-R1- and RCAS-R2-infected embryos. These embryos also show altered dermal development evident in the reduction in density of the developing dermis at HH41. ep, epidermis; de, dermis.
Fig. S2. Western blot analysis of ppf explant extracts cultured in the presence or absence of 25 mM Su5402 for 16 hours. Ppf explants cultured in the presence of 25 mM Su5402 lack detectable di-phosphorylation of ERK1 and ERK2 indicating that the amount of Su5402 used in the experiments presented in Fig. 4 is sufficient to block FGF signaling in vitro. Both antibodies were used at a dilution of 1:5000 in western blot analysis (a-ERK1 and ERK2: Sigma M5670; a-diphosphorylated (dp) ERK1 and ERK2; Sigma M9692). For the preparation of protein extracts explants were washed in PBS and lyzed on ice with kinase buffer (10 mM Tris, pH 7.6, 50 mM NaCl, 1 mM EGTA, 1% Triton X-100, 50 mM NaF, 30 mM sodium pyrophosphate, 10 mM Na3VO4, 2 nM ocadaic acid, and protease inhibitor cocktail; Roche, 1836153), centrifuged at 12,000 g for 10 minutes at 4°C, and the pellet was discarded. Freshly prepared lysates were analyzed by 10% SDS-PAGE and immunoblotted as described previously (Yu and Sato, 1999. J. Cell. Physiol. 178, 235-246).
Fig. S3. Specificity of the aFGF10 antibody. Binding of aFGF10 antibody (Santa Cruz, SC-7375) can only be detected in the first two lanes which show extracts of HH29 embryonic chicken skin and recombinant FGF10 protein, respectively. aFGF10 antibody does not recognize recombinant FGF2-5, FGF7, FGF8 or FGF18. Method: aFGF10 was used at 1:250. 25 mg protein extracted from skin and 50 ng recombinant FGF protein was analyzed by 10% SDS-PAGE and immunoblotted as described previously (Yu and Sato, 1999. J. Cell. Physiol. 178, 235-246). Arrow indicates the position of the FGF10 protein band.
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