Transmission and scanning electron microscopy For transmission electron microscopy, 2 mm3 blocks of full thickness skin were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer. The specimens were then rinsed in 0.1 M cacodylate buffer plus 0.23 M sucrose and 2 mM CaCl2 at pH 7.2, postfixed in 2% osmium tetroxide in 0.1 M cacodylate buffer for 1 h, rinsed in buffer, stained with 2% aqueous uranyl actetate, dehydrated and embedded in Spurr’s resin. 1 mm semi- thin sections were cut with glass knives on a Leica OMU3 ultramicrotome and stained with toluidine blue for light microscopy. Thin sections were cut, stained with lead citrate/ uranyl acetate and visualized with a Hitachi H7000 electron microscope equipped with a SIS Megaview III digital camera. For scanning electron microscopy hairs were placed directly on an aluminium stub and viewed uncoated with a FEI Quanta 200 scanning electron microscope. Supporting Information Figure S1 Analysis of TGase 3 RNA expression upon insertion. Exon 6 encodes the major part of the catalytic center, in particular the cysteine producing the substrate-enzyme intermediate. The selection cassette is inserted at base 790 and introduces a premature stop codon in any transcript formed containing exon 6 at amino acid 245 (murine TGase 3 containing a total of 693 residues). Further, the insertion of the 1600 bp (pgk- Neo-pA) is expected to destabilize this transcript. Should splicing occur around exon 6, not only would this remove the catalytic core region but exon 5 splicing to exons 7, 8, 9, 10 or 11 would generate a frame shift in the transcript. To verify the effect upon the TGM3 transcript, mRNA was isolated from skin, reverse transcribed and assayed at either end of the native message. While the expected DNA fragments, 606 bp (amplified from exons 2 to 5) and 208 bp (exons 7 and 9), were obtained from both wild type and heterozygous skin, neither was seen from mutant skin suggesting instability of the transcript. (TIF) References 1. Aeschlimann D, Thomazy V (2000) Protein crosslinking in assembly and remodelling of extracellular matrices: the role of transglutaminases. Connect Tissue Res 41(1): 1–27. 2. Aeschlimann D, Paulsson M (1994) Transglutaminases: protein cross-linking enzymes in tissues and body fluids. Thromb Haemost 71(4): 402–15. 3. Mycek MJ, Clarke DD, Neidle A, Waelsch H (1959) Amine incorporation into insulin as catalyzed by transglutaminase. Arch Biochem Biophys 84: 528–40. 4. Vader LW, de Ru A, van der Wal Y, Kooy YM, Benckhuijsen W, et al. (2002) Specificity of tissue transglutaminase explains cereal toxicity in celiac disease. J Exp Med 195(5): 643–9. 5. 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Ear (A, E), tongue (B, F), stomach (C, G) and back skin (D, H) (scale bars 100 mm). Lower panel, sections of back skin from TGM32/2 (I–N) and wild type (O–T) mice were incubated with polyclonal antibodies against markers for keratinocyte differentiation. Lor- icrin (I, O), keratin14 (J, P), keratin10 (K, Q), keratin1 (L, R), involucrin (M, S) and filaggrin (N, T) (scale bars 50 mm). (TIF) Figure S3 Analysis of TGase 5 RNA expression upon insertion. To check for any compensatory up-regulation in TGM5 message, total RNA from skin was reverse transcribed by random priming and analyzed by qPCR. Relative expression values (2DDC(t)) for TGM5 were obtained by comparing it with the 18S amplification. No difference was observed between wild type (2.023, sd +/20.411, n = 4) and TGM32/2 animals (2.01, sd +/20.23, n = 4). 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