Olga M. Mazina, Marjorie A. Phillips, Trevor Williams, Carol A

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Redistribution of Transcription Factor AP-2α in Differentiating Cultured Human Epidermal Cells Olga M. Mazina, Marjorie A. Phillips, Trevor Williams, Carol A. Vines, Gary N. Cherr, Robert H. Rice Journal of Investigative Dermatology Volume 117, Issue 4, Pages 864-870 (October 2001) DOI: 10.1046/j.0022-202x.2001.01472.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression of AP-2α, involucrin, and TGM1 mRNA as a function of time. Illustrated at the top are representative phosphorimages of hybridization signals from the mRNA at the times indicated. The bottom two panels show the relative amounts of mRNA normalized to the GAPDH signal for each lane. Each value is the mean of two independent experiments. The vertical arrow at day 8 shows when the cultures reached confluence. Journal of Investigative Dermatology 2001 117, 864-870DOI: (10.1046/j.0022-202x.2001.01472.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 AP-2 DNA binding activity in nuclear extracts. Binding activities were measured using nuclear extracts from cultures harvested on the days indicated. (A) A representative phosphorimage of a resulting gel; (B) relative AP-2 DNA binding activity in nuclear extracts normalized to the binding in the day 4 extract. The asterisk (*) denotes a lane without added extract. Values in the graph represent the means and standard deviations from three independent experiments. The vertical arrow at day 8 shows when the cultures reached confluence. Journal of Investigative Dermatology 2001 117, 864-870DOI: (10.1046/j.0022-202x.2001.01472.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 AP-2 DNA binding activity in cytoplasmic extracts. Binding activities were measured using cytoplasmic extracts from cultures harvested on the days indicated. The asterisk (*) denotes a lane without added extract. NE denotes a lane in which nuclear extract from half-confluent cultures was examined in parallel for comparison. Journal of Investigative Dermatology 2001 117, 864-870DOI: (10.1046/j.0022-202x.2001.01472.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Identity of AP-2 isoforms interacting with the AP-2 response element. Protein complexes were analyzed using nuclear extracts from cultures harvested on the indicated days. Prior to the electrophoresis, incubations were performed in the presence of antibodies specific for the α, β, or γ AP-2 isoforms as shown above the lanes. The asterisk (*) denotes lanes without added extract, and the minus sign (–) denotes samples prepared without added antiserum. Journal of Investigative Dermatology 2001 117, 864-870DOI: (10.1046/j.0022-202x.2001.01472.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Distribution of AP-2α protein between nuclear and cytoplasmic extracts. Nuclear or cytoplasmic extracts (12 µg or 40 µg of protein, respectively) prepared from cultures harvested on the indicated days were resolved by 10% SDS polyacrylamide gel electrophoresis. The relative amounts of AP-2 protein were examined by immunoblotting using a monoclonal antibody specific for the α isoform. Journal of Investigative Dermatology 2001 117, 864-870DOI: (10.1046/j.0022-202x.2001.01472.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 AP-2 localization in cultured hEp. (A) Preconfluent culture showing localization primarily in nuclei. (B) Confluent culture showing localization primarily in nuclei of basal cells. (C) Confluent culture 3 d after medium change, showing diffuse cytoplasmic labeling in the most superficial layer of cells. (D) Confluent culture 8 h after medium change, showing marked nuclear staining in the most superficial layer of cells. (E) Control preconfluent culture processed in parallel with (A) but lacking primary antibody. (F), (G), (H) Same fields as (B), (C), and (D), respectively, counterstained with nuclear-specific BoPro stain. Journal of Investigative Dermatology 2001 117, 864-870DOI: (10.1046/j.0022-202x.2001.01472.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Contrast in localization between hEp, malignant keratinocytes, and 3T3 cells. (A) SCC-9 squamous carcinoma cells, showing predominant nuclear labeling. (B) Postconfluent hEp with almost exclusively cytoplasmic labeling. (C) 3T3 fibroblasts, showing labeling that is cytoplasmic and perinuclear. Journal of Investigative Dermatology 2001 117, 864-870DOI: (10.1046/j.0022-202x.2001.01472.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions