Fig. 2
SCAPER mutation, expression and conservation: a Sanger sequencing demonstrating the SCAPER nonsense mutation: unaffected individual (“homozygous wild-type”), obligatory carrier (“heterozygous carrier”), and an affected individual (“homozygous mutant”). b SCAPER proteomic abundance data generated using the Human Proteome Map (HPM) [32]. c Schematic demonstration of the two SCAPER variants: top panel shows the genomic ___location and exons; middle panel depicts the ___location of the various domains within SCAPER; lower panel highlights the ___location of the p.(L936*) variant, predicted to truncate both transcripts of SCAPER (demonstrated using Ingenuity® Variant Analysis™ isoforms viewed on gene views). d, e Western blot analysis of SCAPER protein in HeLa cells transfected with either wild type (pCBF-FLAG-SCAPER), mutant FLAG-tagged SCAPER (pCBF-FLAG-mutSCAPER) or mock plasmid (pCBF-FLAG), stained using polyclonal anti-SCAPER (d) or anti-FLAG (e) antibodies. Mutant SCAPER protein was observed ~50 kDa smaller than the wild type. These findings were consistent when using either anti-FLAG or anti-SCAPER antibodies. f Although the transfection protocol was identical for all constructs, densitometry for wild-type SCAPER and its mutant counterpart (normalized to total lane protein) showed marked tenfold decrease in mutant protein level. The experiments (d, e) were repeated twice (biological replicates; ex.1, ex.2). Protein quantification data (f) integrate data from ex.1 and ex.2
