1. Localization of Calcineurin/NFAT in Human Skin and Psoriasis and Inhibition of Calcineurin/NFAT Activation in Human Keratinocytes by Cyclosporin A 
Wael I. Al-Daraji, Karen R. Grant, Kerri Ryan, Angela Saxton, Nick J. Reynolds 
Journal of Investigative Dermatology 
Volume 118, Issue 5, Pages (May 2002)
DOI: /j x
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Cyclophilin A, FKBP12, calcineurin B, and NFAT1 are expressed by cultured human keratinocytes. Cell lysates were prepared from cultured keratinocytes, separated by SDS polyacrylamide gel electrophoresis, and immuno blotted with antibodies against cyclophilin A (CyPA), calcineurin B (CaNB), FKBP12, or NFAT1, as indicated. Results from two samples derived from independent donors (lanes 1, 2) are shown. The relative molecular mass of protein markers is indicated to the left of the blots.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Localization of cyclophilin A, FKBP12, calcineurin B, and NFAT1 in normal human skin, lesional psoriatic skin, and nonlesional psoriatic skin.(A) Frozen sections of normal human skin, lesional (plaque) psoriatic skin, and nonlesional (uninvolved) psoriatic skin were immunostained with antibodies against cyclophilin A, calcineurin B, FKBP12, or NFAT1, as indicated. Ni-diaminobenzidine was used as the chromagen (blue/black) and the sections were counterstained with methyl green. (B) Higher power view of NFAT1 immunostaining of normal human skin, nonlesional psoriatic skin, and lesional psoriatic skin showing increased suprabasal nuclear NFAT1 localization in lesional and nonlesional psoriatic skin compared to normal skin. Arrowheads indicate positive nuclear immunostaining. Nonimmune rabbit serum, at a similar dilution to NFAT1, was included in the immunostaining protocol as a negative control. Methyl green counterstaining is seen but no significant blue/black staining is present. Scale bar: 50 µM.
Journal of Investigative Dermatology  , DOI: ( /j x)
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4. Figure 3
Langerhans cells in human skin express cyclophilin A, FKBP12, and calcineurin B. (A) Cyclophilin A immunostaining of normal human skin. (B) Calcineurin B immunostaining of nonlesional psoriatic skin. Expression of cyclophilin A (A) and calcineurin B (B) by Langerhans cells is indicated by arrows. (C), (D), (E) Double-labeling of normal human skin with antibodies to CD1a (Oregon Green 488) (C) and FKBP12 (Alexa 568) (D), visualized by scanning confocal microscopy. Images were overlayed (E). Colocalization of CD1a and FKBP12 appears yellow and is indicated by arrows. The position of the epidermal-dermal junction is indicated by the dashed line. Mid-section images are shown. Scale bar: 50 µM.
Journal of Investigative Dermatology  , DOI: ( /j x)
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5. Figure 4
Nuclear translocation of NFAT1 (A) and calcineurin A and calcineurin B (B) in human keratinocytes is inhibited by cyclosporin A. Human keratinocytes were cultured on coverslips in low calcium MCDB-153 medium (control) and then treated with dimethyl sulfoxide (DMSO) (vehicle control) or TPA (50 nM) plus ionomycin (1 µM) or ↑[Ca2+]o (raised extracellular calcium, 1.5 mM) for 4 h. Some coverslip cultures were pretreated with cyclosporin A (CsA) 1 µM for 1 h, as indicated. Cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Immunofluorescence labeling was performed as described in Materials and Methods. In (A) cells were labeled with anti-NFAT1 antibody (Oregon Green 488) and propidium iodide (PI) and in (B) cells were double-labeled with anticalcineurin B (Alexa 568), and anticalcineurin A (Oregon Green 488). Cells were then visualized using confocal microscopy. The images shown are mid-cell sections. These results are representative of three experiments on keratinocytes derived from three independent donors. Scale bar: 25 µM.
Journal of Investigative Dermatology  , DOI: ( /j x)
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6. Figure 5
Time course of NFAT1 (A) and calcineurin B (B) nuclear translocation in human keratinocytes in response to TPA (50 nM), TPA plus ionomycin (1 µM), and raised extracellular calcium ([Ca2+]o, 1.5 mM). Human keratinocytes were cultured on coverslips and immunostained as described in the legend to Figure 3. The number of cells showing positive nuclear staining was counted using epifluorescence microscopy. At least 225 cells from three independent experiments were assessed at each time point.
Journal of Investigative Dermatology  , DOI: ( /j x)
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7. Figure 6
Translocation of NFAT1-GFP in human keratinocytes. Human keratinocytes were cultured on coverslips in low calcium MCDB-153 medium and then transfected with HA-NFAT1-GFP. Twenty-four hours later cells were visualized by confocal microscopy (A) and then treated with TPA (50 nM) plus ionomycin (1 µM) at 37°C. Re-scanning of the same cell after 1 h (B) showed translocation of HA-NFAT1-GFP to the nucleus. Scale bar: 20 µM.
Journal of Investigative Dermatology  , DOI: ( /j x)
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8. Figure 7
TPA and ionomycin do not induce nuclear translocation of NFAT1 in human dermal fibroblasts. Human dermal fibroblasts were cultured on coverslips in DMEM and 10% fetal bovine serum and then treated with DMSO (vehicle control), TPA (50 nM) plus ionomycin (1 µM), ionomycin (1 µM), CsA (1 µM), or tacrolimus (1 µM) for the indicated times. Cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Immuno fluorescence labeling was performed with anti-NFAT1 antibody (Oregon Green 488) and propidium iodide (PI) as described in Materials and Methods. Cells were then visualized using confocal microscopy. These results are representative of three experiments on fibroblasts derived from three independent donors. Scale bar: 25 µM.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions