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cge12056-sup-0001-AppendixS1.pdfPDF document118KAppendix S1. An expanded methods section.
cge12056-sup-0002-TableS1.pdfPDF document64KTable S1. Desmosomal gene sequence variants in individuals with disease associated mutations in the desmoplakin gene.
cge12056-sup-0003-FigureS1.pdfPDF document662KFig. S1. (a) WB of keratinocyte protein extracts from WT controls and a homozygous (II-1, Carvajal syndrome) and a heterozygous carrier (I-2) of the DSP-p.S2594PfsX8 mutation. Compared to Fig. 1c in the printed manuscript, a threefold amount of protein is loaded in each lane. Small amounts of mutant C-terminal truncated DSP protein are present in the heterozygous mutation carrier. Enhanced chemiluminescence was used for sensitive detection of eventual truncated DSP proteins. (b) Quantification of both DSP isoforms. The homozygous DSP-p.S2594PfsX8 mutation carrier expressed truncated DSP protein isoforms only. Mean values from triplicate WB experiments are shown. The DSP-WT group represents DSP protein expression in seven different WT samples, error bars = group mean ±SD. (c, d, e) Quantification of DSP1/ACTB, DSP1+2/ACTB and DSP1/DSP1+2 protein expression ratios by WB of keratinocytes from family 2, 3, and 4 and WT controls. Mean values from triplicate WB experiments are shown. Error bars indicate group mean ± SD. (f) Western blot including samples of family 2. Compared to Fig. 3d in the printed manuscript, a threefold amount of protein is loaded in each lane. Enhanced chemiluminescence was used for sensitive detection of eventual truncated DSP proteins.
cge12056-sup-0004-FigureS2.pdfPDF document289KFig. S2 (a, b,c) qRT-PCR data from family 2. Error bars indicate mean ± SD of triplicate experiments with keratinocyte pellets from three independent cell cultures. (d) Post-read plot from a TaqMan allelic discrimination SNP assay with fluorescent probes annealing to the WT DSP sequence (X-axis) or the mutant c.3805C>T DSP sequence (Y-axis). Genomic DNA (circles) and cDNA (squares) from DSP-WT (filled symbols) and heterozygous mutation carriers (open symbols) were used in the PCR reaction. A sample without DNA (X) was included to illustrate background fluorescence. The signal from the VIC-probe was significantly reduced in cDNA samples of mutation carriers compared to the corresponding heterozygous genomic DNA samples. (e) Sequence electropherograms with DSP nucleotide sequences containing the 3805 position. Thymidine nucleotide was only present in trace amounts in cDNA from heterozygous mutation carriers (*).
cge12056-sup-0005-FigureS3.pdfPDF document129KFig. S3 (a) DSP-haplotypes of samples used for quantification of the DSP-R1738Q variant protein; p. = paternal allele, m. = maternal allele. (B) Mass spectrometry chromatograms of DSP peptides identified by nLC-MS in keratinocyte protein extracts from samples with different DSP genotypes. (c) Quantification of the DSP-R1738Q variant peptide. Keratinocytes of the digenic DSP-p.K324_E325del/PKP2-p.T526M proband (3.II-3) expressed significantly lower amounts of the DSP-R1738Q variant compared with the healthy DSP-p.K324_E325del carrier (3.II-4), in which the expression of DSP-R1738Q protein was similar to the expression in an individual homozygous for the DSP-p.R1738Q variant. (d) Estimation of DSPK324_ E325del protein. Based on the values in (c), it was estimated that in the proband (3.II-3) approximately 20% of expressed DSP in cytoskeletal protein fraction consisted of mutant DSP-K324_E325del protein, while 3.II-4 apparently degraded the mutant protein and expressed DSP-WT protein only; het. = heterozygous, hom. = homozygous. Error bars = mean ± SD.
cge12056-sup-0006-FigureS4.pdfPDF document2461KFig. S4 (a) IHC of myocardial and epidermal tissue in family 1 with a DSP1 and DSP1+2 antibodies. DSP expression appeared to be normal in the homozygous DSP-p.S2594PfsX8 mutation carrier with Carvajal syndrome whereas the heterozygous mutation carrier had reduced epidermal DSP expression compared with a wild type control. Scale bar = 40 µm. (b) IHC of myocardial and epidermal tissue in family 2 with DSP1 and DSP1+2. The DSP-p.R1269X mutation was associated with reduced epidermal and myocardial DSP expression. Scale bar = 20 µm.
cge12056-sup-0007-FigureS5.pdfPDF document1617KFig. S5 (b) IHC of myocardial and epidermal tissue in family 3 with a DSP1 and PKP2 antibody. Epidermal PKP2 expression was very low but ducts of the adnexal glands stained positive (bottom panel). (b) IHC of myocardial and epidermal tissue in family 4 with DSP1+2 and DSG2 antibodies. The immunoreactive pattern in mutation carriers was indistinguishable from controls.

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