Comparison and analysis of human and chicken <i>Dab1</i> genomic DNA sequences.

<p>Sequences spanning chicken <i>Dab1</i> intron 9 to intron 9B and human <i>Dab1</i> intron 9 to intron 9C were aligned and compared. Intronic regions are in small case whereas exonic regions are shaded and in uppercase. Splice donor (gt) and acceptor (ag) sites are indicated in bold. Both human and chicken <i>Dab1</i> have an exon 9B; human <i>Dab1</i> has an additional exon 9C. TCAT sequences and corresponding Nova binding elements (UCAU in pre-mRNA) are boxed. Multiple Nova binding elements are found upstream of exon 9B (intron 9) in human and chicken <i>Dab1</i> and exon 9C (intron 9B) in human <i>Dab1</i>.</p

Sequence alignment of P1/P4-amplified Dab1 fragments.

<p>HuDab1-E, huDab1-L and chDab1 nucleotide and amino acid sequences are as indicated. The Dab1-L-specific 105 nt two-exon (7/8) region is indicated in blue while the huDab1-E-specific 51 nt exon 9B (57 nt in the case of chDab1-E) is indicated in red. SFK (Y185 and Y198) and Abl/Crk (Y220 and Y232) recognition motifs are boxed. The six italicized nucleotides were only present in a subset of the products analysed.</p

Identification of <i>Dab1</i> splice forms expressed in RB and NB cell lines.

<p>(<b>A</b>) Schematic representation of Dab1 protein showing the region encompassing exons 7 and 8 which contains SFK and Abl/Crk recognition motifs and exons 9, 9B and 9C. The P1 and P4 primers used for the RT-PCR analysis span the entire exon exclusion/inclusion region. (<b>B</b>) DNA amplified in RB cell lines (left panel) and NB cell lines (right panel) using primer set P1/P4. Sizes of amplified DNAs are as indicated and sequences are shown in <b>D</b>. Amplification of actin cDNA was used as a control to ensure that similar amounts of cDNA were used for each cell line. (<b>C</b>) Northern blot analysis of <i>Dab1</i> in RB cell lines. Actin served as the loading control. (<b>D</b>) Alignment of Dab1 sequences corresponding to <i>Dab1</i> splice forms expressed in RB and NB cell lines using primer set P1/P4. The human <i>Dab1</i> genomic intron/exon structure spanning exon 6 to exon 10 is schematically represented. The amino acid sequence encoded by the 530, 481, 430, 376 and 325 bp DNA fragments amplified in <b>B</b> are aligned to show exon inclusion/exclusion in each <i>Dab1</i> splice form.</p

Immunofluorescence analysis of Dab1 (red) and phospho-SFK^Y416 (green) in the developing human retina.

<p>Sections were double-stained with anti-Dab1 and anti-phospho-SFK^Y416 (pSFK) antibodies followed by Alexa 555-conjugated goat anti-rabbit and Alexa 488-conjugated donkey anti-mouse secondary antibodies. Sections were counterstained with the fluorescent dye Hoescht 33258 to label the nuclei. Retinal tissue sections were prepared from human fetal retina at 8 wks gestation (<b>A</b>) and 13 wks gestation (<b>B</b>). The strong staining observed in the RPE is caused by autofluorescence. Abbreviations are: RPE, retinal pigment epithelium; INBL, inner neuroblastic layer; NFL, nerve fiber layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.</p

Co-staining of Dab1 and specific retinal lineage markers at 13 wks gestation.

<p>Retinal tissue sections were double-stained with antibodies to Dab1 and Islet1 (marker of ganglion cells), and Dab1 and AP2α (marker of amacrine cells). Cells that are co-stained with anti-Dab1 and AP2α are indicated by the arrows. Abbreviations are: RPE, retinal pigment epithelium; INL, inner nuclear layer; GCL, ganglion cell layer.</p

RT-PCR analysis of Dab1 deletion and insertion regions.

<p>(<b>A</b>) cDNAs synthesized from poly(A)⁺ RNA from human fetal brain (8 wks gestation), retina (8 wks gestation) and chick retina (E5) were amplified using P1 and P2 primers for deletion analysis and P3 and P4 primers for insertion analysis. Sizes of amplified bands are indicated. (<b>B</b>) Schematic representation of human Dab1-E and Dab1-L proteins and relative positions of primers used for RT-PCR amplification.</p

Analysis of CR-50-treated huDab1-L-expressing chick retinal cells.

<p>GFP-huDab1-L and phospho-SFK⁴¹⁶ (pSFK) expression in mock and CR-50-treated cells. Sixteen hours after DNA transfection, cells were washed and exposed to two sequential treatments of 1 µl/ml CR-50 at 24 h per treatment. Cells were fixed with 4% paraformaldehyde, permeabilized and immunostained with anti-phosphoSFK^Y416 antibody followed by Alexa 555-conjugated goat anti-mouse secondary antibody. The GFP signal in transfected cells was detected by epifluorescence. (<b>A</b>) High magnification view of transfected cells. (<b>B</b>) Low magnification view of transfected cells using the Tile-scan function of the LSM program. A 3×3 tile was used to construct each image. Similar results were obtained in two different experiments.</p

Western blot analysis of RB (A) and NB (B) cell lines.

<p>Fifty µg of protein lysates from each of the indicated tumor cell lines were electrophoresed through a SDS- polyacrylamide gel (10% polyacrylamide for Dab1, actin, Nova1, Nova2; 8% (low BIS) for Reelin) and electroblotted onto nitrocellulose or PVDF membranes. Blots were immunostained with rabbit anti-Dab1 (1∶2000), rabbit anti-Nova1 (1∶1000), goat anti-Nova2 (1∶1500), mouse anti-Reelin (1∶500) or mouse anti-actin (1∶300,000) antibodies, followed by labeling with appropriate HRP-conjugated secondary antibodies. Arrowhead, asterisk and arrow point to full-length Reelin (3428 amino acids), N-R6 Reelin (N-terminal to end of repeat 6) and N-R2 Reelin (N-terminal to end of repeat 2), respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028579#pone.0028579-Jossin1" target="_blank">[48]</a>.</p

Co-staining of Dab1 and specific retinal lineage markers at 8 wks gestation.

<p>Retinal tissue sections were double-stained with antibodies to Dab1 and Islet1 (marker of ganglion cells), Dab1 and MIB-1 (proliferation marker), and Dab1 and AP2α (marker of amacrine cells). Cells that are co-stained with anti-Dab1 and MIB1, and anti-Dab1 and AP2α are indicated by the arrows. Insets show higher magnification of double-stained cells. Abbreviations are: RPE, retinal pigment epithelium; INBL, inner neuroblastic layer; NFL, nerve fiber layer; GCL, ganglion cell layer.</p

Comparison of <i>Dab1</i> splice forms expressed in two primary RB tumors and corresponding cell lines.

<p>cDNAs derived from RB tumors and cell lines were amplified using primer set P1/P4. The sizes of the amplified bands are as indicated.</p