Figure 2

Expression of EFNB1 transcript and protein in primary patient fibroblasts harbouring heterozygous nonsense mutation c.196C > T/p.R66X and heterozygous frameshift mutation c.614_615delCT. (A) The mutation c.196C > T/p.R66X has been shown by direct sequencing of the cDNA. Nucleotide exchange C > T (indicated by an arrow) creates a premature termination codon TGA in exon 2. (B) Wild type and mutant EFNB1 RNA were expressed in a patient fibroblast culture (lane mut). In the control (lane wt) only wild type EFNB1 RNA is expressed. Wild type and mutant transcripts were distinguished by RT-PCR followed by cleavage with restriction enzyme AvaI. Wild type allele is indicated by arrows, mutant allele is indicated by an arrowhead. In lane Ø the RT-PCR product prior to cleavage is shown. (C) Direct sequencing of EFNB1 cDNA of a control (upper panel) and mutation c.614_615delCT (lower panel). Deletion of the CT dinucleotide creates a premature termination codon TGA in exon 4 (indicated by arrows). (D) Wild type and mutant EFNB1 RNA were expressed in a patient fibroblast culture (lane Er4). In the control (lane Er6) only wild type EFNB1 RNA is expressed. Wild type and mutant transcripts were determined by RT-PCR followed by cleavage with restriction enzyme HinfI. Wild type allele is indicated by arrows, mutant allele is indicated by an arrowhead. Size markers are shown in lane M (100 bp DNA ladder, Invitrogen). (E) Western blot analysis of ephrin-B1 expression in lysates of patient fibroblast cultures. Er4 and Er6 show an approximately 50 kDa protein (indicated by an arrow). No smaller truncated protein corresponding to c.614_615delCT was detected at the predicted molecular weight of ≈20 kDa in Er4 (expected size indicated by an arrowhead) using an anti-ephrin-B1 antibody. Protein sizes were determined using Precision Plus Protein™ Standards Dual Color (BIO-RAD).