1. New Function for NF1 Tumor Suppressor
Jussi Koivunen, Heli Ylä-Outinen, Timo Korkiamäki, Seija-Liisa Karvonen, Minna Pöyhönen, Matti Laato, Jaakko Karvonen, Sirkku Peltonen, Juha Peltonen 
Journal of Investigative Dermatology 
Volume 114, Issue 3, Pages (March 2000)
DOI: /j x
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Expression of NF1 tumor suppressor displays marked alteration in cultured keratinocytes in response to elevated external calcium concentration. Cultured normal keratinocytes were fixed in methanol and immunolabeled for NF1 tumor suppressor with antibody NF1GRP(D). The specimens were photographed using the same exposure times and prints were reproduced under identical conditions. (A) Cells cultured in low calcium concentration (<0.1 mM). Note the positive, moderately intense, immunoreaction perinuclearly and in the cell periphery apparently in association with fibrillar structures in some cells. (B) Cells were maintained in high calcium concentration (1.8 mM) for 4 h prior to labeling. The highly intense immunoreaction is associated with finger-like projections extending to the cell periphery in some cells. (C) Cells maintained in high calcium concentration for 8 h prior to labeling. Only a minority of the cells in the monolayer and one large cell on top of the original monolayer display intensely positive labeling for NF1 protein. (D) Control reaction 1: antibody preincubated with appropriate peptide originally used for immunization. Cells were cultured in a high calcium concentration for 4 h. (E) Control reaction 2: antibody preincubated with inappropriate peptide. Cells were cultured in a high calcium concentration for 4 h. Scale bars: 20 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
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3. Figure 2
Double IIF labeling demonstrates association of NF1 tumor suppressor with cytoskeletal intermediate filaments. Keratinocytes maintained in a high calcium concentration for 4 h were fixed in methanol and double immunolabeled with antibodies to CK14 and NF1 (antibody NF1GRP(D)). The same 0.5 μm optical sections were analyzed with CLSM. (A) NF1 protein shows intense, highly organized immunoreaction in one of the differentiating keratinocytes. (B) CK14 immunolabel visualizes cytokeratin-containing intermediate filaments and verifies all cells as keratinocytes. (C) Combined picture shows NF1 in the red channel and CK14 in the green. The yellow color represents a mixture of the two immunosignals indicating that the two proteins are closely located. Note also the apparently nuclear labeling for NF1 protein. Scale bar: 20 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
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4. Figure 3
The presence of NF1 tumor suppressor in keratinocytes extracted with CSK buffer demonstrates high-affinity association between intermediate filaments and NF1 protein. (A-C) Keratinocytes were maintained in a high calcium concentration for 4 h prior to extraction, and were subsequently fixed with methanol and immunolabeled in situ. (A) Immunolabeling for CK14 demonstrating the presence of intermediate filaments in keratinocytes after CSK buffer extraction. (B) Immunolabeling for α-tubulin attests the specificity of the extraction and shows expected negative immunosignal. (C) Immunolabeling with NF1GRP(D) antibody demonstrates NF1 protein in association with intermediate filament cytoskeleton after CSK buffer extraction demonstrating a high-affinity interaction between intermediate cytoskeleton and NF1 protein. (D) Western blotting of the cells treated with CSK buffer and separated into two fractions by centrifugation. The insoluble fraction (intermediate filaments and associated proteins) and the soluble fraction (all other cellular proteins) were analyzed separately. Keratinocytes maintained in low or high calcium concentration for 4–24 h were used for analysis. Antibody NF1GRP(D) demonstrated the presence of a 250 kDa NF1 band, which was abolished by preincubation of the antibody with an appropriate synthetic peptide. Scale bar: 20 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
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5. Figure 4
Multiple antibodies detect NF1 protein on intermediate filaments. Keratinocytes were maintained in a high (1.8 mM) external calcium concentration for 4 h prior to labeling. (A) IIF with antiserum NF1as159, which recognizes a peptide sequence specific for alternatively spliced exon 48a. In analogy to antibody NF1GRP(D), NF1as159 labeling is demonstrated in association with bundles of cytoskeletal filaments. Cells were fixed with methanol. (B) IIF with affinity-purified antibody NF1ab67, which recognizes a peptide sequence specific for alternatively spliced exon 23a. Positive immunoreaction with antibody NF1ab67 was detectable only when the cells were treated with CSK buffer prior to fixation and immunolabeling. Immunoreaction with NF1ab67 was similar but not identical to that found with NF1GRP(D) or NF1as159. Note the beaded labeling pattern of NF1ab67. Scale bars: 20 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
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6. Figure 5
Double IIF labeling demonstrates that NF1 is associated with cytoskeleton during the formation of cellular junctions. Cells were maintained in a high external calcium concentration for 4 h and the same 0.5 μm optical sections were subsequently analyzed with CLSM. NF1 (antibody NF1GRP(D)) is seen in red, desmoplakin or β4 integrin in green. The yellow color represents a mixture of the two immunosignals indicating that the two proteins are closely located. Cells marked 1–6 in frames (A)-(B) represent different stages of cell-cell contact formation. (A) Cell 1: high degree of colocalization of NF1 protein and desmoplakin apparently in close contact with cytokeratin bundles; cells 2–3: reduced degree of colocalization of NF1 protein and desmoplakin. (B) Cell 4: NF1 protein on cytoskeletal filaments and desmoplakin on cell-cell junctions are seen separately; cells 5–6: reduced amount of NF1 immunoreactivity and prevailing desmoplakin immunosignal at the cell-cell contact sites (arrows). (C) NF1 protein and β4 integrin display occasional colocalization in plaques facing the cell culture substratum (arrows). Note also the apparently nuclear labeling for NF1 protein (cells 1, 2, and 4). Scale bars: 20 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
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7. Figure 6
Immunoelectron microscopy demonstrates NF1 protein on bundles of intermediate filaments in normal human epidermis. Immunoelectron microscopy was carried out using antibody NF1GRP(D). The graphs show parts of basal keratinocytes. (A) Note the immunogold particles representative of NF1 protein adjacent to bundles of intermediate filaments near, but not on, a desmosome (D). (B) Immunogold particles are seen near, but not on, hemidesmosomes (HD). BM, basement membrane. Scale bars: 200 nm.
Journal of Investigative Dermatology  , DOI: ( /j x)
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8. Figure 7
Keratinocytes cultured from NF1 patients display highly variable cell size compared with controls. Toluidine blue staining of confluent keratinocyte cultures (A-B). IIF labeling of cultured NF1 keratinocytes for NF1 tumor suppressor, antibody NF1GRP(D) (C). Keratinocytes were maintained for 4 h in high calcium concentration. (A) Keratinocytes cultured from a healthy control person. Note the regular morphology and size of the cells. (B) Keratinocytes cultured from a patient with NF1 display variation in cell size and shape. (C) CLSM image of NF1 keratinocytes maintained for 4 h in a high calcium concentration. Note the low number of NF1-positive cytoskeletal filaments compared with controls in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5. Scale bars: (A, B) 50 μm; (C) 10 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
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