The Expression of Vitamin D-Upregulated Protein 1 in Skin and its Interaction with Sciellin in Cultured Keratinocytes Marie-France Champliaud, Alain.

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The Expression of Vitamin D-Upregulated Protein 1 in Skin and its Interaction with Sciellin in Cultured Keratinocytes Marie-France Champliaud, Alain Viel, Howard P. Baden, MD Journal of Investigative Dermatology Volume 121, Issue 4, Pages 781-785 (October 2003) DOI: 10.1046/j.1523-1747.2003.12539.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression of VDUP1 and sciellin in normal human epidermis and cultured keratinocytes. (a) Lysis buffer extract of human epidermis (lane 1), NM1 cells (lane 2), and HaCat cells (lane 3) showed bands of identical mobility using VD4. Lane 4 was a repeat of lane 1, but without primary antibody. (b) Western blot of protein adherent and eluted from an affinity VD4 column and reacted with VD4 (lane 1) and SC4 (lane 2). Lane 3 was a western blot of the initial extract of HaCat cells reacted with SC4. (c) Western blot of flow through volume of normal rabbit γ-globulin column showed bands with SC4 (lane 1) and VD4 (lane 2) and the elution volume (lane 3) when reacted with SC4 showed no band. (d) Western blot using 34D11 of protein extracted from protein G–agarose beads reacted with the cultured cell lysate and VD4 (lane 1) and the extraction from protein G–agarose beads reacted with lysate and normal rabbit γ-globulin (lane 2). The horizontal lines were standards of 50 and 75 kDa. Journal of Investigative Dermatology 2003 121, 781-785DOI: (10.1046/j.1523-1747.2003.12539.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 VDUP1 was localized to the basal layer of the epidermis but was more widely distributed in the hair follicle. Tissue sections were double-stained with 34D11 and VD4. (a) VDUP1 (red) was present primarily in the basal layer of epidermis, whereas sciellin (green) was in the granular layer. (b) In the infundibulum/isthmus of the follicle, VDUP1 was primarily in the periphery of the ORS. (c) Deeper in a tangential cut follicle, the whole ORS contained VDUP1 and sciellin lined its inner most layer. The cornified IRS was weakly stained. (d) A cross-section in the lower third of the follicle showed staining of the ORS by VDUP1, where as sciellin was restricted to the ORS inner most surface (arrowhead) with evidence of colocalization. The IRS stained for VDUP1 and sciellin with colocalization most evident in its inner most layer (arrow). (e) The bulb region showed that the IRS but not the matrix cells surrounding the dermal papilla (DP) were stained by VD4. The large white dots outline the DP and the small ones, the ORS. Bar, 30 μm. Journal of Investigative Dermatology 2003 121, 781-785DOI: (10.1046/j.1523-1747.2003.12539.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 In lesional psoriatic skin VDUP1 was present throughout the epidermis. Double staining with VD4 (a) and 34D11 (b) showed a broader distribution of the antigens than in normal skin. Overlay (c) of a,b showed that cells expressing sciellin contained high levels of VDUP1. Bar, 30 μm. Journal of Investigative Dermatology 2003 121, 781-785DOI: (10.1046/j.1523-1747.2003.12539.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Differentiation of cultured keratinocytes resulted in colocalization and enhanced expression of VDUP1 and sciellin. Cells were double-stained with 34D11 and VD4. In a–c, the cells were cultured in defined keratinocyte medium and enhanced expression of VDUP1 (a) and sciellin (b) was seen in the differentiating round cells detaching from the dishes. Overlay (c) of a,b confirmed the colocalization of the proteins in the detaching cells (*). In d–f cells were grown in complete medium containing fetal bovine serum and Ca2+ to induce differentiation and were visualized by confocal microscopy to better document coexpression. The differentiated cells expressed higher levels of VDUP1 (d) and sciellin (e) and the overlay (f) of d,e confirmed the coexpression of VDUP1 and sciellin in some cells (*). Bar, 10 μm. Journal of Investigative Dermatology 2003 121, 781-785DOI: (10.1046/j.1523-1747.2003.12539.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions