LEDGF/DFS70, a Major Autoantigen of Atopic Dermatitis, Is a Component of Keratohyalin Granules Kazumitsu Sugiura, Yoshinao Muro, Yuji Nishizawa, Miyako.

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LEDGF/DFS70, a Major Autoantigen of Atopic Dermatitis, Is a Component of Keratohyalin Granules Kazumitsu Sugiura, Yoshinao Muro, Yuji Nishizawa, Miyako Okamoto, Toshimichi Shinohara, Yasushi Tomita, Jiro Usukura Journal of Investigative Dermatology Volume 127, Issue 1, Pages 75-80 (January 2007) DOI: 10.1038/sj.jid.5700487 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Specificity of the LEDGF/DFS70 IDPS70 antisera and immunocytochemistry of thin sections of the epidermis with antisera to LEDGF/DFS70. (a) Protein blot analyses of (a) HeLa cell extracts and (b) HaCaT cell extracts stained with: lane 1, commercial antibodies to LEDGF/DFS70; lane 2, polyclonal S-S antiserum to LEDGF/DFS70; lane 3, polyclonal No. 6369 antiserum to LEDGF/DFS70; and lane 4, preimmune serum of No. 6369. Numbers on the left are molecular weight markers. Thin sections of the epidermis immunostained with (c) anti-LEDGF antiserum No. 6369 or (d) control preimmune antiserum No. 6369. LEDGF/DFS70 was visualized with horseradish peroxidase. Bar=50μm. Journal of Investigative Dermatology 2007 127, 75-80DOI: (10.1038/sj.jid.5700487) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Immunocytochemistry of thin sections of multilayered cultured keratinocytes with an antiserum to LEDGF/DFS70. Thin sections of LabCyte EPI-Model were stained with (a) hematoxylin and eosin, and were immunostained with (b) antiserum No. 6369, and with (c) preimmune serum of No. 6369. Asterisk shows the layer corresponding to the granular layer. Bar=50μm. Journal of Investigative Dermatology 2007 127, 75-80DOI: (10.1038/sj.jid.5700487) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Immunocytochemistry of thin sections of the epidermis with anti-Bip/GRP78 antibodies. Thin sections of the epidermis were immunostained with polyclonal anti-Bip/GRP78 antibodies. Arrow points to the basement membrane. Bip/GRP78 was visualized with horseradish peroxidase. Bar=50μm. Journal of Investigative Dermatology 2007 127, 75-80DOI: (10.1038/sj.jid.5700487) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Localization of LEDGF/DFS70 in the KGs by immunoelectron microscopy analysis. The subcellular localization of LEDGF/DGS70 in the keratinocytes of the granular layer and stratum corneum were determined by immunoelectron microscopy. LEDGF/DFS70 was found in KGs, (a–c) in the granular layers, and (e) in the nuclei. (d) In controls, the antiserum No. 6369 pre-incubated with the immunizing peptide was used for immunoelectron microscopy. (b) Magnified image of the inset in (a). Arrow indicates the irregularly shaped and multivesicular structures of the KGs. An arrowhead points to a keratin bundle. The K4M embedding method was used. Goat anti-rabbit antiserum coupled to 10nm immunogold particles was used for detection. Bar=200nm. Journal of Investigative Dermatology 2007 127, 75-80DOI: (10.1038/sj.jid.5700487) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Colocalization of LEDGF/DFS70 and profilaggrin in the KGs of cells in the granular layer. A double immunostaining method was used with anti-LEDGF/DFS70 atniserum and anti-filaggrin antibody. Profilaggrin, the precursor of filaggrin, is a marker of KGs. Five nanometer immunogold-conjugated secondary antibody was used for LEDGF/DFS70 detection and 10nm gold for filaggrin immunostaining. (a) The inset picture is a magnified picture of square. (b) Photograph showing the same pattern of immunostaining in the stratum corneum. Bar=(a) 200nm and (b) 500nm. Journal of Investigative Dermatology 2007 127, 75-80DOI: (10.1038/sj.jid.5700487) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Bip/GRP78 localizes in KGs and in the stratum corneum. (a, b) Double immunostaining method was used with anti-LEDGF/DFS70 antiserum and anti-Bip/GRP78 antibody. Five nanometer immunogold conjugated second antiserum was used for LEDGF/DFS70 and 10nm for Bip/GRP78. (a) A photograph showing KGs, and (b) a photograph showing the staining pattern in the stratum corneum. Bar=(a) 200nm and (b) 500nm. Journal of Investigative Dermatology 2007 127, 75-80DOI: (10.1038/sj.jid.5700487) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions