Isoform-Specific Differences in the Size of Desmosomal Cadherin/Catenin Complexes  Leslie J. Bannon, Betty L. Cabrera, Kathleen J. Green  Journal of Investigative Dermatology  Volume 117, Issue 5, Pages 1302-1306 (November 2001) DOI: 10.1046/j.1523-1747.2001.01512.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Immunoprecipitation analysis of stoichiometric ratios among desmoglein/plakoglobin complexes. In order to analyze the stoichiometry of complexes formed by the various desmosomal cadherin isoforms and plakoglobin, stable L-cell lines were established expressing these components, each with a C-terminal myc tag (a).(b) Four lines expressing Dsg2 and plakoglobin, 743.M37, 743.J26, 743.B7, and 743.A1, were analyzed by immunoprecipitation with anti-6D8 anti-Dsg2 antibody, and immunoblot with 9E10 anti-myc antibody to detect C-terminal myc-epitope tags. A cell line expressing Dsg1 and Dsg2 with plakoglobin, 3299.C5 was subjected to immunoprecipitation with 982 anti-Dsg1 antibody, and 6D8 anti-Dsg2 antibody, followed by immunoblot with 9E10. Four lines expressing EcadDsg3 and plakoglobin, 790.2, 790.3, 790.4, and 790.12 were analyzed by immunoprecipitation with DECMA anti-E-cadherin antibody, and immunoblot with 9E10. (c) Stable lines expressing no ectopic proteins (control), Dsg1 and plakoglobin (Dsg.Pg19), or Dsg2 and plakoglobin (A6L) were metabolically labeled for 3 h and subjected to immunoprecipitation with 982 or 6D8, respectively. Gels were transferred to nitrocellulose and exposed to film (35S). These blots were subsequently rehydrated and subjected to 9E10 anti-myc immunoblot (myc). Journal of Investigative Dermatology 2001 117, 1302-1306DOI: (10.1046/j.1523-1747.2001.01512.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 The molecular size of Dsg1.Pg complexes exceeds that of Dsg2.Pg or EcadDsg3.Pg complexes. Lysates prepared from L-cells stably expressing C-terminally myc-tagged Dsg1 and plakoglobin (PG), Dsg2 and PG, or EcadDsg3 and PG were subjected to analysis by gel filtration chromatography and sucrose gradient sedimentation. (a) Lines Dsg.Pg19 and 790.14 were lyzed in 1% TTBS and subjected to sequential gel filtration chromatographic runs over a Superose 6 column. Fractions were analyzed by immunoblot using antibodies against myc-tagged PG (polyclonal 1407, which recognizes both PG and the myc-epitope tag), or against the myc tag (polyclonal pmyc). The peak maximum of Dsg1.Pg occurred at fraction 23, whereas the peak maximum of EcadDsg3.Pg occurred at 24.5. (b) The elution profile of Dsg1.Pg complexes (triangle) is plotted against the elution profile of EcadDsg3.Pg complexes (diamond). (c) Line 3299.C5, stably expressing C-terminally myc tagged Dsg1, Dsg2, and PG, was lyzed in 1% OTBS, and subjected to gel filtration. Fractions were analyzed by immunoblot using antibodies directed against Dsg1 (18D4), Dsg2 (6D8), or plakoglobin (1407). The peak maximum of Dsg1 occurred at f22.5, whereas the peak maximum of Dsg2 occurred at f23.5. (d) The elution profile of Dsg1 (triangle) is plotted vs the elution profile of Dsg2 (diamond). In the above experiments, the column was standardized with (T) thyroglobulin molecular weight 669 kDa, (F) ferritin 440 kDa, (C) catalase 232 kDa, and (A) aldolase 158 kDa. (e) Lines DsgPg19 and 790.14 were lyzed and mixed prior to layering over a 10–20% sucrose gradient, then subjected to ultracentrifugation. Migration of complexes through the gradient was detected by immunoblot using 18D4 or a polyclonal antibody directed against E-cadherin (795). The center of the peak for Dsg1 occurred at f13, and peaks occurred for EcadDsg3 at f10 and f14. (f) The migration of Dsg1.Pg complexes vs EcadDsg3.Pg complexes is plotted. Journal of Investigative Dermatology 2001 117, 1302-1306DOI: (10.1046/j.1523-1747.2001.01512.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions