Functioning Methionine-S-Sulfoxide Reductases A and B Are Present in Human Skin  Karin U. Schallreuter  Journal of Investigative Dermatology  Volume 126, Issue 5, Pages 947-949 (May 2006) DOI: 10.1038/sj.jid.5700086 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 MSRA and MSRB are both expressed throughout the human epidermis. (a) In situ immunofluorescence of methionine sulfoxide reductases A and B shows partial co-localization of the two enzymes. Cryostat sections (5 μm) of human full skin biopsies were fixed in ice-cold methanol (6 min), rehydrated in phosphate-buffered saline (PBS) for 30 s, and blocked with normal donkey serum (NDS, 10% in PBS) for 90 min, then washed in PBS (5 min). Subsequently, sections were incubated overnight at 4 °C with the primary antibody (monoclonal anti-MSRB; mouse, 1:50 in 1% NDS; Autogen Bioclear UK Ltd., Calne, UK), washed 3× with PBS, incubated with FITC-labeled secondary antibody (donkey anti-mouse IgG, 1:100; Jackson ImmunoResearch Ltd., Soham, UK) for 60 min, washed 4× with PBS and once with PBS/Tween, blocked with NDS (10% in PBS) for 90 min, and then washed once in PBS for 5min. MSRA was visualized using polyclonal anti-MSRA antibody (rabbit, 1:50 in 1% NDS; Autogen Bioclear UK Ltd, Calne, UK) for 3h at RT, followed by a 3× wash in PBS, and was incubated with TRITC-labeled secondary antibody (donkey anti-rabbit IgG, 1:100; Jackson ImmunoResearch Ltd., Soham, UK) for 60 min, and then washed 4× with PBS and once with PBS/Tween. Slides were embedded in Vectashield Mounting Medium with 4′,6-diamino-2-phenylindole (Vector, Peterborough, UK). Fluorescence pictures were taken with a Nikon Eclipse 80i microscope equipped with ACT-2U software (Nikon UK, Kingston, UK). (b) Western blotting confirmed the presence of MSRB (13kDa calculated) in epidermal undifferentiated and differentiated keratinocyte cellular extracts. Proteins from primary epidermal undifferentiated and differentiated keratinocyte cell cultures were separated by SDS-PAGE (10%), transferred onto a polyvinylidene difluoride membrane, and incubated with the anti-MSRB antibody (1:2,000, overnight at 4 °C), followed by peroxidase-labeled antibody (goat anti-mouse IgG, 1:5,000, 1h at RT; Sigma, Poole, UK). Bands were detected by ECL (Sigma, Poole, UK, and Eastman Kodak, Rochester, New York) at the expected size of 13kDa. Journal of Investigative Dermatology 2006 126, 947-949DOI: (10.1038/sj.jid.5700086) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions