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1.
Figure 2

Figure 2. Perp is internalized with DSG3 and PG upon PV IgG exposure. From: Loss of the Desmosomal Protein Perp Enhances the Phenotypic Effects of Pemphigus Vulgaris Autoantibodies.

(a) Representative immunofluorescence images of Perp in human keratinocyte monolayers treated with NL (left) or PV (right) IgG for 0, 3, or 6 hours. (b–d) Representative immunofluorescence images of Perp and DSG3, PG, or DP in human keratinocyte monolayers treated with PV IgG for 0, 3, or 6 hours. Nuclei are stained with DAPI. Scale bar = 10 μm and applies to all photomicrographs in the figure.

Bichchau Nguyen, et al. J Invest Dermatol. ;129(7):1710-1718.
2.
Figure 3

Figure 3. Perp is internalized into endosomes in a similar pattern to PG upon PV IgG exposure. From: Loss of the Desmosomal Protein Perp Enhances the Phenotypic Effects of Pemphigus Vulgaris Autoantibodies.

Representative immunofluorescence images for the endosome marker EEA-1 and either Perp or PG in human keratinocyte monolayers treated with PV IgG for 0 (a) or 3 hours (b). Nuclei are stained with DAPI. Arrows indicate several puncta in which Perp or PG colocalizes with EEA-1. Scale bar = 10 μm and applies to all photomicrographs in the figure.

Bichchau Nguyen, et al. J Invest Dermatol. ;129(7):1710-1718.
3.
Figure 4

Figure 4. Perp enters lysosomes in response to PV IgG exposure. From: Loss of the Desmosomal Protein Perp Enhances the Phenotypic Effects of Pemphigus Vulgaris Autoantibodies.

Representative immunofluorescence images for the lysosome marker CD63 and either Perp or PG in human keratinocyte monolayers treated with PV IgG for 0 (a) or 3 hours (b). Nuclei are stained with DAPI. Arrows indicate several puncta in which Perp or PG colocalizes with CD63. Scale bar = 10 μm and applies to all photomicrographs in the figure.

Bichchau Nguyen, et al. J Invest Dermatol. ;129(7):1710-1718.
4.
Figure 6

Figure 6. Loss of Perp enhances the intercellular adhesion defects induced by PV IgG. From: Loss of the Desmosomal Protein Perp Enhances the Phenotypic Effects of Pemphigus Vulgaris Autoantibodies.

A mechanical dissociation assay on wild-type and Perp−/− mouse keratinocyte monolayers treated with normal (NL) or PV IgG for 24 hours was performed, and the numbers of resulting cell monolayer fragments per 40 × field were counted. Results represent the mean±SEM (n = 12). Statistical significance (*) was determined using the unpaired Student’s t-test (P<0.05).

Bichchau Nguyen, et al. J Invest Dermatol. ;129(7):1710-1718.
5.
Figure 5

Figure 5. Loss of Perp cooperates with PV serum to induce depletion of DSG3 and PG. From: Loss of the Desmosomal Protein Perp Enhances the Phenotypic Effects of Pemphigus Vulgaris Autoantibodies.

Western blot analysis of Triton X-100 and urea fractions from wild-type (WT) and Perp−/− mouse keratinocyte monolayers treated with normal (NL) or PV serum (PV) for 24 hours. An examination of Perp solubility and levels as well as the effects of Perp loss on the solubility profiles of key desmosomal proteins (DP, DSG3, and PG) is shown. GAPDH and Keratin 14 (K14) serve as loading controls for the Triton-soluble and urea fractions, respectively.

Bichchau Nguyen, et al. J Invest Dermatol. ;129(7):1710-1718.
6.
Figure 1

Figure 1. Characterization of PV IgG. From: Loss of the Desmosomal Protein Perp Enhances the Phenotypic Effects of Pemphigus Vulgaris Autoantibodies.

Studies in (a), (b), and (c) were conducted using human keratinocytes cultured in 0.5mm calcium for 18 hours before experimentation. (a) Representative immunofluorescence images show that PV IgG, but not normal (NL) IgG, recognizes a plasma membrane antigen with a localization pattern similar to that of DSG3. Scale bar = 10 μm. (b) Western blot analysis examining reactivity of human sera and antibodies against human keratinocyte protein lysates. PV and NL sera, purified PV and NL IgG fractions, and DSG3 antibodies were tested. (c) Immunoprecipitation–Western blot analysis of proteins immunoprecipitated by NL and PV IgG and probed for the desmosome components DSG3, PG, and DP. 10% of the input is shown. (d) Mechanical dissociation assay performed on monolayers of wild-type mouse keratinocytes treated with PV IgG or NL IgG for 24 hours. The numbers of fragments generated were counted in four 40 × fields in each of three experiments, and results represent the mean±SEM (n = 12). Statistical significance (*) was determined using the unpaired Student’s t-test (P<0.05).

Bichchau Nguyen, et al. J Invest Dermatol. ;129(7):1710-1718.

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