1. Expression of FcRn, the MHC Class I-Related Receptor for IgG, in Human Keratinocytes 
Karla Cauza, Gabriele Hinterhuber, Ruth Dingelmaier-Hovorka, Karin Brugger, Gabriele Klosner, Reinhard Horvat, Klaus Wolff, Dagmar Foedinger 
Journal of Investigative Dermatology 
Volume 124, Issue 1, Pages (January 2005)
DOI: /j X x
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Expression of neonatal Fc receptor (FcRn) α-chain mRNA. (A) After reverse transcription of RNA from human keratinocytes (obtained from two different cell strains and of skin samples from two different donors), PCR was performed with primers specific for human FcRn α-chain. Amplified PCR products were run on 1.2% agarose gel and stained with ethidium bromide. Control represents a PCR sample running without cDNA. (B) Nucleotide sequence of 457 bp as detected by direct sequence analysis of PCR product is shown. The sequence is identical to the respective part of known human FcRn sequence (GenBank AF ). The primers are indicated by lowercase letters. (C) RNA from human keratinocytes of same samples as in (A) was hybridized with FcRn DIG-labeled antisense probe. Northern blot analysis revealed a specific product at about 1.5 kb for FcRn α-chain in all epidermal keratinocytes tested.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Detection of human neonatal Fc receptor (FcRn) α-chain by immunoblotting and immunoprecipitation. (A) Total cellular proteins from cultured keratinocytes were resolved on a 12% SDS-PAGE gel under reducing conditions. When immunoblotting was performed with rabbit anti-FcRn α2-extracellular domain antibody (Ab), a reactive band was detected at 46 kDa representing FcRn α-chain as shown in lane 2. The same reactivity was obtained by immunoblotting with goat anti-FcRn cytoplasmic tail Ab (lane 3). When immunoblotting was performed with irrelevant rabbit and goat IgG no reactivity was found (lanes 4 and 5) Lane 1 shows the molecular weight marker. (B) Immunoprecipitation was performed using the rabbit anti-FcRn α2-extracellular domain Ab, covalently bound to immobilized sepharose. After PAGE and transfer to nitrocellulose, immunoblotting with the rabbit anti-FcRn α2-extracellular domain Ab resulted in a single reactive band at 46 kDa (lane 2) indicating FcRn α-chain expression. Identical reactivity was obtained when the strip of the same probe was incubated with goat anti-FcRn cytoplasmic tail Ab (lane 6). Additionally, when anti-β2 microglobulin (β2m) Ab was used for immunoblotting this probe a single band at about 12 kDa was seen showing coprecipitated β2m (lane 3). No reactivity was seen with control rabbit and goat IgG used for immunoblotting (lanes 4 and 5) and after cross-linking of irrelevant rabbit IgG with consecutive immunoblotting using the rabbit anti-FcRn α2-extracellular domain Ab (lane 7). Lane 1 shows the molecular weight marker.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Indirect immunofluorescence on human keratinocytes. (A) Cultured keratinocytes were fixed, permeabilized with Triton X-100, and incubated with rabbit anti-neonatal Fc receptor α2-extracellular domain antibody (Ab). After exposure to Alexa 488-conjugated donkey anti-rabbit IgG Ab laser microscopic evaluation showed a distinct granular–vesicular fluorescence pattern throughout the cell. (B) Alexa 488-conjugated donkey anti-rabbit IgG Ab without first Ab gave no specific fluorescence pattern, Scale bar: 10 μm.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Cellular distribution of neonatal Fc receptor (FcRn) on human keratinocytes. (A) The obtained histogram by fluorescence-activated cell sorting (FACS) analysis using biotinylated second step as bridge antibody (Ab) on fixed, non-permeabilized keratinocytes for surface detection of FcRn shows a slight shift in fluorescence intensity (red) compared with control stain with irrelevant rabbit IgG (black), indicating a weak expression of FcRn α-chain on the cell surface of human keratinocytes. (B) FACS analysis on fixed and permeabilized cells for detection of intracellular localized FcRn performed without a bridge Ab revealed a prominent shift in the fluorescence intensity (red) compared with the control stain with irrelevant rabbit IgG (black). Histograms are given with ordinate indicating the number of cells and abscissa indicating the fluorescence intensity.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
IgG-neonatal Fc receptor (FcRn) binding is pH dependent. Human IgG cross-linked to Protein L was incubated with keratinocyte lysates at pH 6. Eluted proteins were run on 12% SDS gel, transferred to nitrocellulose, and immunoblotted with rabbit anti-FcRn α2-extracellular domain antibody (Ab) (lane 2) and goat anti-FcRn cytoplasmic tail Ab (lane 3) showing a specific reactivity at 46 kDa for FcRn (lanes 2 and 3). To show that IgG–FcRn interaction could be blocked by Fc fragments the incubation of Protein L IgG was performed at pH 6.0 in the presence of 500 μg per mL Fc fragments, and consecutive immunoblotting of precipitated antigens with anti-FcRn α2-extracellular domain Ab (lane 4) and anti-FcRn cytoplasmic tail Ab (lane 5) gave no reactivity. For control, lysates and IgG were incubated in the presence of F(ab′)2 fragments at pH 6.0, and immunoblotting with anti-FcRn α2-extracellular domain Ab (lane 6) and anti-FcRn cytoplasmic tail Ab (lane 7) show specific reactivity at 46 kDa. When human IgG Protein L beads were incubated with NP-40 keratinocytes lysates at pH 7.4, no specific reactivity was seen after the eluted proteins were run on 12% SDS gel, transferred to nitrocellulose and immunoblotted with rabbit anti-FcRn α2-extracellular domain Ab (lane 8), and goat anti-FcRn cytoplasmic tail Ab (lane 9). Lane 1 shows the molecular weight marker.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Neonatal Fc receptor (FcRn) expression in normal human epidermis. (A) For immunohistochemical detection, the avidin–peroxidase method and rabbit anti-FcRn α2-extracellular domain antibody were used. FcRn gives a distinct granular–vesicular staining throughout the keratinocytes predominantly of the basal, but also from the suprabasal cell layer. (B) Irrelevant rabbit IgG shows no specific labeling pattern. Scale bar: 20 μm.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions