Genetic Modifiers of MeCP2 Figure 1. MeCP2 alleles used to generate transgenic Drosophila: protein expression, phosphorylation at serine 423, and association with polytene chromosomes. A. Four MECP2 alleles were cloned into pUAST to generate transgenic flies. The methyl-CpG-binding domain (MBD) is represented by blue boxes and the transcription repression domain (TRD) is represented by green boxes. The nuclear localization signal (NLS) falls within the TRD. B. Western blot analysis demonstrates expression of each of the alleles when driven by GMR-Gal4. Two distinct MeCP2 antibodies were utilized in order to recognize each allele to confirm that a deletion removed an epitope region. C. Immunoblot with a phospho-specific antibody shows phosphorylation in the three alleles retaining amino acid S423. D. Immunoblot with the phospho-specific MeCP2 S423 antibody in negative control, extracts from MeCP2 expressing flies when treated with calf intestinal phosphatase and untreated MeCP2 extracts. The treated samples fail to produce a band with the phospho-specific antibody, but demonstrate MeCP2 expression with the whole MeCP2 antibody (E-I’’). Immunoflourescence of squashed polytene chromosomes dissected from 3rd instar larvae raised at 25uC. Control larvae do not have MeCP2 immunoreactivity (E-E’’). All MeCP2 alleles demonstrate accumulation of the MeCP2 protein in banded pattern along the polytene chromosomes (F-I’’). doi:10.1371/journal.pgen.1000179.g001 PLoS Genetics | www.plosgenetics.org 3 September 2008 | Volume 4 | Issue 9 | e1000179