Fig 1: NV848 restores SBDS protein expression in lymphoblastoid cell lines from SDS patients. (a), representative experiment; LCL from UPN58 or from healthy donors (Control) were incubated with ataluren (2.5 µM) or analogues (10 µM each), or vehicle alone (DMSO) for 24 h. SBDS protein expression was quantified by Western blot analysis. (b), Densitometry analysis of bands as shown in panel (a). Data are mean ± SEM of five independent experiments conducted on LCL obtained from patients UPN26, UPN58, UPN75, UPN82, and UPN106. (c), PBMC from UPN108 were incubated with ataluren (2.5 µM) or NV848 (10 µM), or vehicle alone (DMSO) for 72 h. SBDS protein expression was quantified by Western blot. (d), Densitometry of bands depicted in panel (c); data are mean ± SEM of three independent experiments. Student’s t test for paired data has been calculated and indicated within the histograms.
Fig 2: Additional studies of the genomic segment that includes SBDS supports the presence of an insertion of unknown origin within the paternal allele. a. Agarose gel analysis of a 13.1 kb long-range PCR product spanning SBDS and flanking region, the region targeted by the Southern blotting studies (Figures 2 and 3). DNA from patient (BAB3762), parents (BAB3763- mother, BAB3764-father) and a normal controls (CTL1 and CTL2) were amplified using primers DelFb + KpnR, and then digested with either KpnI or SacI. KpnI did not digest the 13.1 kb PCR product, consistent with lack of amplification of the paternal allele. Consistently, SacI digestion of the 13.1 kb PCR product showed an identical pattern in samples and controls. b. Sanger sequencing of intron 2 amplified along with exon 2 using short range PCR revealed inconsistent segregation of a paternal genotype for two polymorphic SNPs in the patient (BAB3762). *Non-digested PCR product.
Fig 3: NV848 improves Granulocyte-macrophage colony forming units in BM-MNC isolated from SDS patients. BM-MNC from a cohort of 16 SDS patients were isolated and colony assays were performed. Cells were incubated in the presence of ataluren (Ata, 2.5 µM) or analogues (10 µM each). Granulocyte-macrophage colony forming units (CFU-GM) and burst forming units-erythroid (BFU-E) were counted after 7 days (a), 14 days (b) and 21 days (c) of incubation. Data are mean ± SEM of 16 independent experiments. Student’s t-test for paired data has been calculated and indicated within the histograms.
Fig 4: Southern blotting of SBDS and SBDSP1 in samples from family HOU1479 using Xba I restriction enzyme.
Fig 5: Approximate location of the inherited SV at the SBDS locus based on Southern blotting assays using KpnI and XbaI restriction enzymes. a. Size and location of the expected segments obtained using KpnI and XbaI restriction enzymes based on the reference genome (blue arrows) and extra segments obtained exclusively in SV carriers (BAB3762 and BAB3764 - purple arrows). The nature of the SV is currently unknown although the Southern blotting results are consistent with a genomic insertion of at least 2.8 kb (represented by a crosshatch red box) somewhere between SBDS 5’flanking region and intron 3 (represented by a red box with an arrow on top). Red box: Southern blotting probe. Yellow rectangle highlights a common probe target region between the reference genome and the genome of the SV carriers. b. An alternative hypothesis of SV present within the pseudogene. Genomic coordinates as of GRCh37/ hg19 to SBDS and SBDSP1 loci are represented on top of each region. CEN: centromere; TEL: telomere.
Supplier Page from Abcam for Anti-SBDS antibody [EPR7820]