1. Characterization of Mouse Profilaggrin: Evidence for Nuclear Engulfment and Translocation of the Profilaggrin B-Domain during Epidermal Differentiation 
Dan Zhang, Seetha Karunaratne, Monica Kessler, Donna Mahony, Joseph A. Rothnagel, Dr. 
Journal of Investigative Dermatology 
Volume 119, Issue 4, Pages (October 2002)
DOI: /j x
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Schematic showing the organization of the profilaggrin gene and the protein coding domains. The domains are denoted after the terminology ofPresland et al (1997) as follows: (a) calcium-binding domain; (b) B-domain; (c) partial filaggrin peptides; (d) filaggrin repeats, and (e) C-terminal hydrophobic domain. Two forms of filaggrin repeat sequences have been identified based on their length of 255 and 250 residues (765/750 bp), which are denoted as types A and B, respectively (seeRothnagel and Steinert, 1990). The gene contains nine type B repeats and three type A repeats.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Sequence of the 5′ end of the mouse profilaggrin gene. The sequence shows the 5′ end of the transcription unit, the 5′ noncoding region (exon 1), two introns, and the first 554 amino acids. The TATA box, OctB, Sp1, ets/jen, AP1, NF-κB, Dlx3/OctA, and intron splice sites are indicated. The transcription initiation site is indicated by a bent arrow. The sequences of exons 1, 2, and 3 are in uppercase. The promoter region and introns are in lowercase. The conceptual translation of the open reading frame is shown below the nucleotide sequence. The furin recognition sequence demarcating the junction between the N-terminal leader sequence and the partial filaggrin repeat is indicated by shaded lettering (residues 280–283). The hydrophobic residues of the first filaggrin linker are highlighted (residues 455–462). The B-domain (residues 202–217) and filaggrin repeat (residues 328–343) peptide sequences used to produce antibodies are underlined.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Comparison of the N-terminal calcium-binding domain of mouse profilaggrin with human profilaggrin and related proteins. The conceptual translated N-terminal residues of mouse profilaggrin (mFlg) are compared with human profilaggrin (FLG) (Markova et al, 1992;Presland et al, 1992), human trichohyalin (THH) (Lee et al, 1993), mouse hornerin (mHorn) (Makino et al, 2001), and mouse repetin (mRptn) (Krieg et al, 1997). The conserved helix-turn-helix motifs of the EF-hands (Lewit-Bentley and Rety, 2000) are indicated. The sequences encoding the two EF-hands are separated by an intron whose position is conserved (indicated by an arrowhead). The sequences were aligned using CLUSTALW.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Sequence comparison of mouse and human pro filaggrin B-domains. The conceptually translated B-domain sequences were aligned using BLAST2. Identical residues are indicated with dark shading and conserved nonidentical residues with light shading. The nuclear localization signals were identified using PSORTII and are indicated by an asterisk. The furin protease recognition sequence that demarcates the C-terminal end of the B-domain (Presland et al, 1997) is underlined.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Double-label immunofluorescence analysis of B-domain and filaggrin expression in 5-d-old mouse skin and in mouse primary keratinocytes.(A) Granular layer and cornified layer cells are double labeled with filaggrin repeat (red) and the B-domain (green) antibodies. Colocalization is observed as yellow. (B) A view of upper granular layer and transition cells double labeled with the B-domain (green) and lamin B (red). (C) A view of upper granular layer and transition cells double labeled with the filaggrin repeat (green) and lamin B (red) antibodies. (D) Enlarged view of the boxed region in (B) showing B-domain staining within a nucleus of an upper granular layer/transition cell. (E) Enlarged view of the boxed region in (C) showing filaggrin repeat staining in the cytoplasm of an upper granular layer/transition cell. (F) A differentiating keratinocyte double labeled with B-domain (green) and nuclear lamin B (red) antibodies. (G) A differentiating keratinocyte double labeled with filaggrin repeat (green) and lamin B (red) antibodies. Note the fine granular appearance of the B-domain and filaggrin staining pattern in (F) and (G). (H) A late-differentiating keratinocyte labeled for the B-domain (green) and lamin B (red). (I) A late-differentiating keratinocyte double labeled for the filaggrin repeat (green) and lamin B (red) antibodies. Note the larger aggregates of B-domain and filaggrin staining in (H) and (I) and the B-domain staining in and around the nucleus and the cytoplasmic localization of the filaggrin repeat. CL, cornified layer; GL, granular layer; TL, transition layer. Scale bar: 10 µm.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Immunoelectron microscopy analysis of B-domain and filaggrin expression. Ultrathin sections of a 5-d-old mouse backskin epidermis showing the expression of filaggrin repeat (10 nm gold, pseudocolored red) and the B-domain (5 nm gold, pseudocolored green). (A) A section showing a transition cell (TC) and a neighboring cornified layer cell (CL) above it and a granular layer cell (GC) below it. (B) The boxed area in (A) enlarged to show B-domain labeling associated with nuclear remnants and filaggrin repeat labeling over filaments. (C), (D) Two different upper granular layer cells labeled for the B-domain. Note the keratohyalin granule (KG) being engulfed by the nucleus in (D). (E) A close up of the keratohyalin granule in (D) showing B-domain labeling of the granule, which is surrounded by an apparently intact nuclear membrane. (F) A section stained with uranyl acetate and lead citrate showing a granular layer cell with a deeply indented nucleus. CL, cornified layer; GC, granular layer cell; KG, keratohyalin granule; NUC, nucleus; NM, nuclear membrane; NR, nuclear remnants; PM, plasma membrane; TC, transition cell. Scale bar: 100 nm.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions