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Thomas E. Stout, Trevor McFarland, John C

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1 Recombinant Filaggrin Is Internalized and Processed to Correct Filaggrin Deficiency 
Thomas E. Stout, Trevor McFarland, John C. Mitchell, Binoy Appukuttan, J. Timothy Stout  Journal of Investigative Dermatology  Volume 134, Issue 2, Pages (February 2014) DOI: /jid Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Immunocytochemistry of HEK-293Ts after filaggrin (FLG) application. Immunocytochemistry using a Trx probe after an application of 8 μg of mFLG-RMR for 1.5 hours, 8 μg of mFLG+RMR for 1.5 hours, 80 μg of mFLG+RMR for 1.5 hours, or 80 μg of mFLG+RMR for 4.5 hours. Protein concentration was 0.4 μg μl−1. Bar=50 μm. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Immunocytochemistry (ICC) and western blots of human dermal fibroblasts (HDFs). (a) ICC using filaggrin (red) and cytoskeletal phalloidin (green) antibodies after an application of 5 μg of mFLG+RMR-Trx for 4 hours, 5 μg of mFLG-RMR for 4 hours, or medium only for 4 hours. Protein concentration was 0.04 μg μl−1. Bar=50 μm. Western blot using a FLG probe against HDF cells treated with 20 μg of either (b) mFLG+RMR-Trx or (c) mFLG-RMR for 4 hours. Applied filaggrin (red arrow) and β-actin (black arrow) are highlighted. Protein concentration was 0.01 μg μl−1. (d) Tandem MS/MS spectral analysis of three subcellular marker proteins: fatty acid synthase (cytoplasmic), Na+/K+ ATPase subunit alpha-1 (membrane), and poly(ADP-ribose) polymerase (nuclear). Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Immunofluorescence of reconstructed human epidermis (RHE) after recombinant filaggrin application. Immunohistochemistry using a mouse-specific filaggrin probe after an application of 50 μg of mFLG+RMR for 15 minutes, 50 μg of mFLG+RMR for 60 minutes, or 50 μg of mFLG+RMR for 240 minutes. Protein concentration was 0.5 μg μl−1. Bar=50 μm. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Immunofluorescence of flaky tail and C57/BL6 dorsal skin. Immunohistochemistry using filaggrin (FLG) antibodies against (a) adult flaky tail mice treated with 50 μg of mFLG+RMR (0.5 μg μl−1), (b) adult flaky tail mice treated with a vehicle solution only, or (c) untreated adult C57/BL6 mice. Bar=100 μm. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Western blot of mouse tissue. Western blot of adult flaky tail dorsal tissue treated with 50 μg of mFLG+RMR (0.5 μg μl−1, 1 hour), adult flaky tail dorsal tissue treated with 50 μg of mFLG-RMR (0.5 μg μl−1, 1 hour), adult flaky tail dorsal tissue treated with a vehicle solution only (1 hour), untreated C57/BL6 mice, pure mFLG+RMR protein, or pure mFLG-RMR protein. Wild-type filaggrin monomer (red arrow) and processed monomeric recombinant filaggrin (black arrow) are highlighted. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Immunohistochemistry, histology, and electron microscopy of mouse tail epidermis. (a–c) Immunohistochemistry, (d, e) hematoxylin and eosin staining, and (f–i) environmental-scanning electron microscopy (E-SEM) of (a, d, f, h) p18 flaky tail mice treated with 50 μg of mFLG+RMR (0.5 μg μl−1) daily for 14 days, (b, e, g, i) p18 flaky tail mice treated with a vehicle solution daily for 14 days, or (c) p18 untreated C57/BL6 mice. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

8 Figure 7 Profilaggrin schematic and recombinant mFLG+RMR protein sequence. (a) Schematic of murine profilaggrin with isolated filaggrin monomer linked to 5′-RMR tag. (b) Corresponding sequence of filaggrin (FLG) +RMR recombinant protein. Red, purple, blue, orange, and green amino acids denote the Trx tag, polyhistidine tag, filaggrin monomer, flanking junctional filaggrin sequences, and RMR motif, respectively. UTR, untranslated region. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

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