Proteomic Profiling Identifies an UV-Induced Activation of Cofilin-1 and Destrin in Human Epidermis  Paul J. Hensbergen, Astrid E. Alewijnse, Johanna Kempenaar, Rose C. van der Schors, Crina I.A. Balog, Andre M. Deelder, Gerrit Beumer, Maria Ponec, Cornelis P. Tensen  Journal of Investigative Dermatology  Volume 124, Issue 4, Pages 818-824 (April 2005) DOI: 10.1111/j.0022-202X.2005.23597.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 UV-induced shifts in protein expression profiles of reconstructed human epidermis. (A) Two-dimensional electrophoresis pattern of reconstructed human skin equivalents of control and solar-irradiated protein samples. The proteins were separated on a pH 3–10 non-linear IPG strip followed by a 13% SDS-polyacrylamide gel as described in the methods. The proteins were visualized using silver staining. Marked spots indicate spots that are significantly up- or downregulated after 1 h irradiation (cumulative dose of 3.75 J per cm2) in four independent experiments performed in duplicate. Encircled spots indicate spots that were regulated in both silver-stained and Coomassie-stained gels. The boxed spots indicate spots that could not be identified in Coomassie-stained gels. (B) Relative spot density in control (white bars) or irradiated samples (black bars). The four bars indicate the four independent epidermal cultures and experiments. In case a spot is absent in a gel this is denoted by an *. The spots were identified by MALDI-MS and sequence analysis (see Figure S2). Journal of Investigative Dermatology 2005 124, 818-824DOI: (10.1111/j.0022-202X.2005.23597.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Magnified two-dimensional polyacrylamide gel electrophoresis image of control and irradiated reconstructed human skin equivalent of four independent cultures and experiments. The relative density of the spots 5207 and 6206 (as measured with PD-Quest) is indicated in the top left corner of the figure, with the black bars representing the control sample and the white bars representing the sample after solar-simulated irradiation. Journal of Investigative Dermatology 2005 124, 818-824DOI: (10.1111/j.0022-202X.2005.23597.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Effect of 30 or 60 min UV irradiation (cumulative dose of 1.825 and 3.75 J per cm2, respectively) of human skin equivalents on the relative density of spot 5207 (representing phospho-cofilin) and spot 6206 (representing phospho-destrin). Data are given ±SD (four independent cultures) relative to control (no UV irradiation). Asterisks indicate values significantly different from the control (Student' t test; *p<0.05). Journal of Investigative Dermatology 2005 124, 818-824DOI: (10.1111/j.0022-202X.2005.23597.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Effect of UV irradiation on phosphocofilin-1 and phospo-destrin expression in human skin. (A) Effect of 3 and 4 h UV irradiation (cumulative dose of 11.25 and. 15 J per cm2, respectively) of excised human skin on the relative density of phospho-cofilin (spot 5207) and phospho-destrin (spot 6206). The relative density of both spots (as measured with PD Quest) is indicated in the top left corner of the figure with the black bars representing the control sample, the gray bars 3 h UV, and the white bars 4 h UV. Also given are the magnified two-dimensional polyacrylamide gel electrophoresis images of control and irradiated excised human skin of this experiment (representative for three independent donors). (B) Western blot analysis of phospho-cofilin (p-cofilin-1) and total cofilin (T-cofilin-1) demonstrating a decline in phosphorylated cofilin after UV exposure. Human skin equivalents (n=3) or excised skin (ex vivo; n=1) were irradiated with UV for the indicated time points. Subsequently, proteins were extracted, separated on a 10% SDS-PAGE gel, and transferred onto nitrocellulose membranes. Membranes were incubated with a phospho-cofilin-specific antibody and visualized using a chemiluminescence kit. After stripping the same membrane was incubated using a total cofilin specific antibody followed by the appropriate HRP-conjugated secondary antibody and visualized. Journal of Investigative Dermatology 2005 124, 818-824DOI: (10.1111/j.0022-202X.2005.23597.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions