Desmoglein 2 Monoclonal Antibody (6D8)
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Figure 1 No alterations in N-cadherin and beta-catenin after knockdown of Dsg2 in melanoma cells. (A) Immunoblots showing efficient Dsg2 depletion in MeWo and C32 melanoma cells. Equal amounts of proteins were loaded. 1: MeWo, Dsg2 siRNA; 2: MeWo, non-targeting (nt) siRNA; 3: C32, Dsg2 siRNA; 4: C32, nt siRNA. In MeWo, Dsg2 reduction was 7.9-fold three days after Dsg2 siRNA transfection and 5.1-fold six days thereafter when the intensity of the bands was normalized against the GADPH immunoblots serving as loading controls. In C32, Dsg2 was 12.7-fold or 122.8-fold reduced. By contrast, protein amounts of N-cadherin (N-cad) and beta-catenin (beta-cat) were virtually unchanged upon Dsg2 depletion. Molecular weight markers (from top to bottom): Dsg2 immunoblots: 158, 116 and 97.2 kDa (day 3); 212, 158 and 116 kDa (day 6); N-cad immunoblots: 116, 97.2 and 66.4 kDa (day 3); 158, 116 and 97.2 kDa (day 6); beta-cat immunoblots: 158, 116, 97.2 and 66.4 kDa (day 3 and 6); GADPH immunoblots: 55.6, 42.7, 34.6 and 27 kDa (day 3 and 6). (B) Immunofluoresence microscopy of Dsg2-depleted (upper panel) and nt siRNA-treated C32 cells (lower panel), showing virtual absence of Dsg2 three days after knockdown. In cells treated with nt siRNA Dsg2 is accumulated at the cell surface and at cell borders. Antibodies to N-cad and beta-cat react at cell-cell junctions and along cell borders, in patterns unaffected by Dsg2 contents. Bars: 200 um.
Figure 7 Upregulation of SgII and SN upon Dsg2 depletion and increased melanoma cell migration upon stimulation with SN. (A, B) Real time PCR showing significantly increased SgII mRNA in subconfluent but not in confluent Dsg2-depleted MeWo and C32. In subconfluent cultures harvested three days after the first siRNA transfection, 4.1-fold more SgII mRNA (MeWo, A) or 4.4-fold more SgII mRNA (C32, B) was detected after Dsg2 knockdown. However, in confluent cultures harvested after six days amounts of SgII mRNA were equal in the C32 samples (B) and slightly decreased in Dsg2-depleted MeWo (1.3-fold, A). (C) RIA demonstrating marked upregulation of SN in cellular extracts of Dsg2-depleted MeWo (4.42 vs. 2.24 fmol/probe, p = 0.0704) and slight SN increase in Dsg2-depleted C32. Bars: SD; * p<=0.05, *** p<=0.001. (D) Immunoblots of total cell lysates from subconfluent Dsg2-depleted MeWo (lane 1), nt siRNA-treated MeWo (lane 2), Dsg2-depleted C32 (lane 3) and nt siRNA-treated C32 (lane 4), harvested three days after the first siRNA transfection. SgII (detected with rabbit antiserum GTX116446) was upregulated 1.6-fold in Dsg2-depleted MeWo as compared to MeWo controls and 1.5-fold in Dsg2-deleted C32 as compared to C32 controls when the intensity of the bands was normalized against the GADPH blot serving as loading control. Dsg2 was depleted 3.1-fold (MeWo) or 26.3-fold (C32) after knockdown. Molecular weight markers (from top to bottom): SgII immunoblot: 97.2, 66.4 and 55.6 kDa; Dsg2
Figure 8 Immunohistochemistry of SgII and Dsg2 on paraffin sections of primary malignant melanomas and melanoma metastases. (A-C, J-L) Primary nodular malignant melanoma (NMM) with a tumor thickness of 0.8 mm (pT1a; no. 4 in the Table S3 ). (D-F, M-O) Primary NMM of 10 mm thickness (pT4a; no. 1). (G-I) Melanoma metastasis of the mamma (no. 8). (P-R) Cutaneous melanoma metastasis of the temple (no. 15). In the upper part of the figure (A-I), tumors were immunostained with two different SgII antisera (A, D, G: GTX116446 and B, E, H: LS-C39034), or, for control, with secondary goat anti-rabbit HRP-IgG (C, F, I). Clearly positive immunoreactions are seen in the cytoplasm of the melanoma cells with both SgII antisera in all tumors examined. In the lower part (J-R), tumors were reacted with antibodies against Dsg2 (rb5). Diffuse Dsg2-positive reactions are detected in the cytoplasm and/or at the surface of the melanoma cells (J, M, P: overviews; K, N, Q: higher magnifications). In addition, Dsg2 appears to be focally enhanced at the cell-cell contacts of the primary NMM of 10 mm thickness and the cutaneous metastasis (N, Q). Dsg2-positive structures serving as internal positive controls on each slide, i.e. sweat glands (L), basal epidermis (O) and hair follicles (R) are shown on the right hand side of each row. Bars: 50 um (L, Q), 200 um (P) or 100 um (all other micrographs).
Western blot analysis of Desmoglein-2 was performed by loading 30 µg of HEK-293 (lane1), A549 (lane2), A-431 (lane3), and K562 (lane4) cell lysate using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and Pierce™ Power Blotter System (22834). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. Desmoglein-2 was detected at ~122 kDa using Desmoglein-2 Mouse Monoclonal Antibody (Product # 32-6100) at 1-2 µg/mL in 5 % skim milk at 4°C overnight on a rocking platform. Goat Anti-Mouse - HRP Secondary Antibody (Product # 62-6520) at 1:4000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).
Western Blot (WB)
Immunohistochemistry (Paraffin) (IHC (P))
Miscellaneous PubMed (MISC)
Host / Isotype
PBS, pH 7.4
0.1% sodium azide
Desmosomes are cell-cell junctions between epithelial, myocardial, and certain other cell types. Currently, three desmoglein subfamily members have been identified and all are members of the cadherin cell adhesion molecule superfamily. These desmoglein gene family members are located in a cluster on chromosome 18. This protein has been identified as the autoantigen of the autoimmune skin blistering disease pemphigus vulgaris.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.