Autoantibodies in Vitiligo Patients Recognize Multiple Domains of the Melanin- Concentrating Hormone Receptor  Raju V.S.R.K. Gottumukkala, Elizabeth A. Waterman, Laura M. Herd, Philip F. Watson, Anthony P. Weetman, E. Helen Kemp  Journal of Investigative Dermatology  Volume 121, Issue 4, Pages 765-770 (October 2003) DOI: 10.1046/j.1523-1747.2003.12494.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 SDS–PAGE and autoradiography of products arising from in vitro translation of MCHR1 cDNA and its deletion derivatives. MCHR1 cDNA and its deletion derivatives were translated in vitro in a TnT T7 Coupled Reticulocyte Lysate System and the radio-labeled products analyzed by electrophoresis in a 12.5% SDS–polyacrylamide gel followed by autoradiography as described in Materials and Methods. Deletion derivatives of MCHR1 in vitro translated from: pMCHR1-138 (lane 1); pMCHR1-139 (lane 2); pMCHR1-178 (lane 3); pMCHR1-218 (lane 4); pMCHR1-258 (lane 5); pMCHR1-298 (lane 6); pMCHR1-338 (lane 7); pMCHR1-378 (lane 8); pMCHR1 (lane 9). Journal of Investigative Dermatology 2003 121, 765-770DOI: (10.1046/j.1523-1747.2003.12494.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Antibody indices of vitiligo patient sera. Vitiligo and control sera were analyzed in radio-binding assays with radio-labeled ligands encoded by a panel of MCHR1 deletion constructs as detailed in Materials and Methods. The antibody index shown for each serum sample is the mean of at least two experiments. The dotted lines represent the upper level of normal for each radio-binding assay calculated as the mean antibody index +3 SD of a population of 20 healthy controls. Journal of Investigative Dermatology 2003 121, 765-770DOI: (10.1046/j.1523-1747.2003.12494.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 SDS–PAGE and autoradiography of radio-labeled proteins following immunoprecipitation with vitiligo patient sera.In vitro translated radio-labeled proteins were used in radio-binding assays with MCHR1 antibody-positive vitiligo patient as detailed in Materials and Methods prior to analysis by SDS–PAGE. MCHR1 proteins encoded by: pMCHR1 (lane 1) and pMCHR1-378 (lane 2) immunoprecipitated by vitiligo patient serum (V1); pMCHR1-338 (lane 4) and pMCHR1-298 (lane 3) immunoprecipitated by vitiligo patient serum (V2); pMCHR1-258 (lane 5) and pMCHR218 (lane 6) immunoprecipitated by vitiligo patient serum (V3). Journal of Investigative Dermatology 2003 121, 765-770DOI: (10.1046/j.1523-1747.2003.12494.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions