1. Glycine Substitution Mutations Cause Intracellular Accumulation of Collagen XVII and Affect Its Post-Translational Modifications 
Laura Huilaja, Tiina Hurskainen, Helena Autio-Harmainen, Raija Sormunen, Hongmin Tu, Silke C. Hofmann, Taina Pihlajaniemi, Leena Bruckner-Tuderman, Kaisa Tasanen 
Journal of Investigative Dermatology 
Volume 129, Issue 9, Pages (September 2009)
DOI: /jid
Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Immunofluorescence and immunoelectron microscopy analysis of wild-type and mutant collagen XVII in COS-7 cells. COS-7 cells were transiently transfected with pcDNA3 vector coding for wild-type collagen XVII, glycine substitutions G539E, G609D, G612R, G627V, and G633D, and a deletion of amino acids 779–787 (Δ779–787) (for details see Supplementary Information). Culturing and analysis of transfected COS-7 cells using immunoelectron microscopy are described in Supplementary Information. In immunofluorescence staining, localization of wild-type and mutated collagen XVII in transiently transfected COS-7 cells was analyzed with the polyclonal collagen XVII NC16A antibody as described earlier (Huilaja et al., 2008), except that Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes, Leiden, the Netherlands; dilution 1:200) was used as a secondary antibody. The close mutations G609D and G612R, as well as G627V and G633D, were previously shown to alter the collagen XVII structure similarly (Väisänen et al., 2005), and thus only G609D and G627V were studied here. (a) Signal for wild-type collagen XVII (in green) is present in plasma membrane, whereas collagen XVII with glycine substitutions G539E (b), G609D (not shown), and G627V (c) is mostly detected as patchy intracellular staining. The localization of Δ779–787 is similar to that of the wild type (d). Pictures were taken using original magnification x40. In electron microscopy, gold immunolabeling of the wild-type collagen XVII is localized to the plasma membrane (PM) (e). G627V-mutated collagen XVII gold immunolabeling is seen in the endoplasmic reticulum (ER) (f), whereas G539E-mutated collagen gold immunolabeling is detected more in the endoplasmic reticulum (g) and less in the plasma membrane (h). Gold particles were manually calculated from 20 different cells in each group and the data were subjected to Mann–Whitney U-test to justify the results. Bar=200nm.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Immunoblotting and shedding of mutated collagen XVII. (a) The size of the mutated collagen XVII fragments was analyzed after precipitation from cell extract (100μl) and cell culture media (400μl) using 4–15% Ready Gel SDS-PAGE (Bio-Rad, Hercules, CA) and Western blotting with polyclonal NC16A antibody (dilution 1:1,000) (Schumann et al., 2000). In cell extracts, the full-length collagen XVII molecules migrated similarly to the wild type. The glycine-substituted G609D, G612R, G627V, and G633D ectodomains present in culture media migrated slightly more slowly than the wild type. The deletion of amino acids 779–787 and the glycine-substituted G539E ectodomain showed no molecular-weight shift. The substitution V703M appears as a missense mutation control and had no effect on migration. (b) Time-frame experiment of COS-7 cells transfected with the wild-type and mutated collagen XVII. Forty-eight hours after transfection, the cells were surface biotinylated with delta-biotinoyl-ε-aminocaproic acid N-hydroxysuccinimide ester according to the manufacturer's recommendations (Roche, Mannheim, Germany), changed to biotin-free medium, and cultured for 72 hours. After five washes with phosphate-buffered saline, the cells were cultured in fresh medium supplemented with ascorbic acid. The cell extract and the media were collected at regular intervals for 72 hours and processed separately as described by Schäcke et al. (1998). The precipitates were immunoblotted with streptavidin-coupled alkaline phosphatase (Roche) to detect biotinylated collagen XVII (Franzke et al., 2002, 2006). (c) The scanning and quantitation analysis of the immunoblotting signals with Quantity One software (Bio-Rad) showed that the shed ectodomain was clearly detectable after 2 hours in the media of cells transfected with the wild type and Δ779–787, whereas the G539E, G609D, and G627V ectodomains were not notably recovered until after 6 hours.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions