Additional file 1: Table S1. of Novel missense mutation in the bZIP transcription factor, MAF, associated with congenital cataract, developmental delay, seizures and hearing loss (AymĂŠ-Gripp syndrome)

List of reported paediatric cataract genes selected for sequencing in this study. (DOCX 39 kb

Additional file 2: of Novel missense mutation in the bZIP transcription factor, MAF, associated with congenital cataract, developmental delay, seizures and hearing loss (Aymé-Gripp syndrome)

List of  annotated and selected variants. (XLSX 41 kb

Evaluation of p15^INK4B expression by Western immunoblotting.

<p>(A, B) Validation of p15^INK4B antibody specificity by Western blotting using GST-tagged, full length, recombinant human p15^INK4B (rp15^INK4B) and p16^INK4A (rp16^INK4A) proteins. M, molecular weight markers. (A) Reactivity of antibody to rp15^INK4B, where lane 1, 100ng of rp15^INK4B; Lane 2, 10 ng rp15^INK4B; Lane 3, 1 ng rp15^INK4B P; Lane 4, 100 pg rp15^INK4B. Single bands of the expected molecular weights (for protein incorporating GST-tag) are apparent. (B) Reactivity of antibody to rp16^INK4A in order to determine any non-specific binding to the highly related protein p16^INK4A, where lane 1, 100 ng of rp16^INK4A; Lane 2, 10 ng rp16^INK4A; Lane 3, 1 ng rp16^INK4A; Lane 4, 100 pg rp16^INK4A. (C) Rat brain cortex (lane 1), liver (lane 2), optic nerve (lane 3) and retina (lane 4) samples probed for rp15^INK4B. A 15 kD protein is detectable in all tissue samples; results plotted on bar graph to show quantitative distribution. (D) Expression of p15^INK4B in retinal (lanes 1–3) and optic nerve (lanes 4–6) samples. Labelling for β-actin (house-keeping gene product) is also shown; results for p15^INK4B plotted on bar graph to show quantitiative distribution, relative to actin levels.</p

Validation of the p16^INK4A antibody from Santa-Cruz (sc-1661).

<p>(A) Evaluation of the specificity of the p16^INK4A antibody, sc-1661, by Western blotting, using GST-tagged, full length, recombinant, human p16^INK4A protein (rp16^INK4A). Molecular weight markers were used to determine size of detected gel products (A; lane M). Lane 1, 100 ng of rp16^INK4A; Lane 2, 10 ng rp16^INK4A. A single band of the expected molecular weight (including GST-tag) is apparent. (B) Rat brain cortex (lane 1), liver (lane 2), optic nerve (lane 3) and retina (lane 4) samples probed for p16^INK4A protein, using sc-1661. None of the four tissues under investigation unambiguously express p16^INK4A protein. Labelling for β-actin (house-keeping gene product) is also shown. (C) The antibody was further tested against retina (lanes 1–3) and optic nerve (lanes 4–6) samples obtained from three different rats. The presence of p16^INK4A protein is not detectable in any sample. (D, E) In formalin-fixed, paraffin-embedded tissue sections of cervical adenocarcinoma, incubation with sc-1661, identifies so-called ‘block’ or ‘diffuse’ immunolabelling of ductal (D) and epithelial (E) tissue with a high signal-to-background, as compared to adjacent tissue (arrow). Scale bar = 30 µm.</p

Representative images of p14^ARF immunolabelling in human ocular tissues.

<p>In formalin-fixed, paraffin-embedded sections of cervical intraepithelial neoplasia, p14^ARF expression is identified, as expected, within nuclear inclusions (A). In formalin-fixed, paraffin-embedded human eyes, no positive labelling for p14^ARF is discernible in the corneal epithelium (B), trabecular meshwork (TM)/Schlemm’s canal (SC) (C), retina (D), unmyelinated optic nerve (E), or myelinated optic nerve (F). Scale bar = 15 µm. GCL, ganglion cell layer; INL, inner nuclear layer.</p

Evaluation of MTAP expression in ocular tissues.

<p>(A) Evaluation of the MTAP antibody by Western immunoblotting using GST-tagged, full length, recombinant, human MTAP protein (rMTAP). Molecular weight markers were used to determine size of detected gel products (A; lane M). Lane 1, 100 ng rMTAP; Lane 2, 10 ng rMTAP; Lane 3, 1 ng rMTAP; Lane 4, 100 pg rMTAP. A single band of the expected molecular weight (including GST-tag) is apparent; results plotted on bar graph to show quantitative distribution. (B) Rat brain cortex (lane 1), liver (lane 2), optic nerve (lane 3) and retina (lane 4) samples probed for MTAP. A protein is detected at the correct molecular mass for MTAP in the liver, optic nerve and retinal samples. Labelling for β-actin (housekeeping gene) and α-tubulin (neuronal tissue marker) are also shown; results plotted on bar graph to show quantitative distribution. (C) Evaluation of MTAP expression in retina (lanes 1–3) and optic nerve (lanes 4–6) samples obtained from three different rats. MTAP protein is detectable in all samples; results plotted on bar graph to show quantitative distribution. (D–G) Evaluation of MTAP antibodies in malignant pleural mesothelioma. In formalin-fixed, paraffin-embedded tissue sections, many inflammatory cells, as identified by immunoreactivity for CD45 (leukocyte common antigen; D), display positive labelling for MTAP using the Proteintech antibody (PT Ab; E). Some fibroblastic cells, negative for CD45 (F), also display positive labelling for MTAP using the PT antibody (G, arrow). Scale bar = 30 µm.</p

Expression of MTAP mRNA in rat tissues.

<p>C_T, cycle threshold;</p>1<p>ΔCT, target C_T - cyclophilin C_T;</p>2<p>target mRNA level expressed relative to cyclophilin.</p

Distribution of genotypes of the <i>TCF4</i> TGC repeat alleles in FECD cases and controls.

<p>The numbers of individuals with each of the three possible genotypes of the dichotomised repeat alleles are shown. S represents short (<40 repeats; non-expanded) and L long (≥40 repeats; expanded) allele. SS represents homozygous non-expanded, LL homozygous expanded, and SL heterozygous with one non-expanded and one expanded allele.</p

Immunocytochemical localization of p19^ARF protein in cultured, undifferentiated, non-confluent, rodent RGC-5 cells.

<p>(A–C) Use of the Santa Cruz antibody (A), shown in relation to nuclear DAPI counterstaining (B, C), reveals little P19^ARF protein labeling except for some cytoplasmic granules (arrowheads). There is no obvious nuclear labeling. (D–F) Use of the p19^ARF antibody from Upstate, however, denotes clear nuclear labeling within cells. The positive labeling appears to be present in nucleolar-like structures within cell nuclei (as denoted by DAPI-positive staining; arrows). This labeling is present in approximately 75% of cells. Scale bar = 10 µm.</p

Expression of P16^INK4A/P19^ARF mRNA in rat tissues.

<p>C_T, cycle threshold;</p>1<p>ΔC_T, target C_T - cyclophilin C_T;</p>2<p>target mRNA level expressed relative to cyclophilin.</p