Supplementary Figure 7: BIN-67 analysis.

(a) Chromatogram of the splice-site mutation in genomic DNA from allele 1. An asterisk denotes the mutation. (b) Chromatogram of the splice-site mutation in genomic DNA from allele 2. An asterisk denotes the mutation. (c) Effect of the mutation on allele 1. Top, chromatograms of cDNA sequence, retaining the beginning of intron 15 and the end of intron 16. Only one allele, allele 1, is being amplified, as the mutation in allele 2 is not present at the end of intron 16 (asterisk). Bottom, diagram of primer location and the expected size of primers. The entire product including both introns was too large to amplify and is not seen on the gel in e. (d) Effect of the mutation on allele 2. Top, chromatogram of cDNA sequence, splicing out exons 15 and 16. Bottom, diagram of primer location and the expected product size. With two exons spliced out, the sequence is in frame with no amino acid changes but does splice out part of the SNF2_N domain, including the DEXDc domain, which is part of the ATPase domain (Figure 1). (e) Gel of BIN-67 results. Lane 1, GAPDH control. Lanes 2 and 3, PCR product from intronic primers 1 and 2 showing that, for allele 2, although the entire product was too large to amplify, the beginning of exon 16 and end of intron 17 are present. Lane 4, BIN-67 cells treated with cycloheximide and non-treated, showing that no larger fragment degraded by nonsense-mediated decay was recovered with cycloheximide. Lane 6, PCR product from control cDNA, showing the correct product size. S, sense; AS, antisense; CHX, cycloheximide; +Ctrl, positive control; Int, intronic primer.