Electron microscopy of weak transgenic <i>Oa1−/−,</i> Gαi3 (Q204L) mouse RPEs and densitometry analysis of their melanosomes compared to those of wild-type NCrl and Oa1<i>−/−</i> mice.

<p>A. Electron micrograph of mouse RPEs from lines 374, 377, and 396. B. Histogram representing the percent distribution of melanosomal sizes. C. Melanosomal density analysis.</p

A: Identification of <i>Oa1−/−,</i> Gαi3 (Q204L) transgenic mice. The lentivirus construct used to generate <i>Oa1−/−,</i> Gαi3 (Q204L) transgenic mice contains the constitutively active Gαi3 (Q204L) cDNA driven by the <i>Oa1</i> promoter and ires-GFP.

<p>Abbreviations: RSV, Rous sarcoma virus promoter; U3-HIV-R-U5, 5′ and 3′ detailed long terminal repeats (LTRs); psi, packaging signal for viral RNA into virus capsids to continue the infection of HIV in its host; RRE, Rev-responsive element; cPPT, central polypurine tract; <i>Oa1</i> (ocular albinism type 1) promoter; Gαi3 (Q204L), constitutively active Gαi3 cDNA; ires, internal ribosome entry site; GFP, green fluorescence protein; WPRE, Woodchuck hepatitis virus post-transcriptional regulatory element; sin U3, self-inactivating element that relies on the introduction of a deletion in the U3 region of the 3′ (LTR). <i>XbaI,</i> restriction enzyme site. Two short thin black arrows indicate the forward and reverse ires-GFP primers used for PCR amplification. The probe used for Southern blots binds to Gαi3 (Q204L) cDNA at the position indicated in the figure. <b>B:</b> 1.8% agarose gel showing PCR analysis of the <i>Oa1</i> gene in putative founder transgenic mice. Specific primer sets (HPRT and <i>Oa1</i>) were used to amplify a 400 bp band of the HPRT cassette and a 500 bp of the endogenous <i>Oa1</i> gene, respectively. The 20 putative transgenic founders analyzed on the gel had the 400 bp band, indicating that all animals were generated in the <i>Oa1−/−</i> background. <b>C:</b> Identification of positive transgenic founders by PCR. 1.8% agarose gel identifying positive Gαi3 (Q204L) transgenic mice. Genotyping was done using specific primers that amplify a 372 bp fragment between the ires and GFP regions of the transgenic construct. Controls used: <i>Oa1−/−</i>, NCrl, C57Bl/6 genomic DNA samples and transgenic plasmid. The Master mix was loaded as a control for contamination.</p

Southern blot analysis of transgenic progeny.

<p>Positive transgenic mice were identified by the presence of the expected 4.7 µg of mouse genomic DNA. <b>A.</b> Positive transgenic mice presenting a strong signal on the Southern blot: Lane 1: line 142. Lane 2: line 131. Lane 3: line 157. <b>B:</b> Positive transgenic mice with a weak signal: Lane 4: line 377. Lane 5: 374, Lane 6: line 396. <b>C:</b> Mice without integrated transgene show no radioactive signal: Lane 7: line 223. Lane 8: Line 275. Lane 9: line 276. <b>D</b>. Negative controls NCrl and line 13 (Lanes 10 and 11) and positive control transgenic mouse line 16 (Lane 12).</p

GFP expression in the RPE varies in different transgenic mice, suggesting a possible correlation between level of GFP expression and transgene copy number.

<p>A: Western blot using RPE/choroid protein extracts and GFP antibody. Positive transgenic mouse lines 231,175 and 174 expressed GFP in the RPE. Controls NCrl and <i>Oa1−/−</i>, as well as mice without integrated transgene (lines 375 and 223) did not express GFP. B: Confocal images (magnification: 174X) of the RPE from positive transgenic mice show expression of GFP.</p

Electron microscopy and densitometry analysis of the progeny of transgenic founders, comparing the RPEs of strong positive transgenic Gαi3 (Q204L) animals and those of mice without integrated transgene.

<p>A. Electron micrographs of RPEs from positive-strong transgenic mice lines 131, 142, and 157. B. Electron micrographs of RPEs from mice without integrated transgene lines 223, 275, and 276. C and D. Histograms representing the percent of RPE melanosomal size distribution in transgenic mice and animals without integrated transgene, respectively. E and F. Melanosomal density analysis of transgenic and non-transgenic progeny, respectively.</p

Table1. Primer sequences (5′ to 3′) used for PCR and Southern blot genotyping.

<p>Table1. Primer sequences (5′ to 3′) used for PCR and Southern blot genotyping.</p

Far-UV CD spectrum of Zbed4 (black curve) and curve obtained using the CONTINLL program (white curve).

<p>The CONTINLL-calculated curve conforms well to the experimental spectra of Zbed4. SELCON and CDSSTR-calculated curves (not shown) were essentially identical to that of CONTINLL.</p

Co-localization of Zbed4 with ERα and MYH9 in Y79 retinoblastoma cells, carried out as described in Materials and Methods.

<p>The immunostaining results indicate that both ERα and MYH9 co-localize with Zbed4 in these cells. The major difference is that in A co-localization is seen in the nuclei and cytoplasm for Zbed4 and ERα whereas in B it is only observed in the cytoplasm for Zbed4 and MYH9. C. Immunoprecipitation experiments were carried out as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035317#pone-0035317-g007" target="_blank">Figure 7C</a> with Y79 retinoblastoma cells (Input, lanes 1 and 6) but using antibodies against Zbed4 (lanes 1, 2 and 4) or MYH9 (lanes 3, 5 and 6) instead; both Zbed4 and MYH9 are detected in each immunoprecipitate.</p

Nucleotide sequence of DNA fragments selected by the CASTing method.

<p>dsDNA (CASTrandom oligonucleotide, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035317#pone.0035317.s003" target="_blank">Table S3</a>, after synthesis of the complementary strand using PCR-specific primers CAST-F and CAST-R) was incubated with Zbed4 to form protein-DNA complexes that were immunoprecipitated using Zbed4 antibody and protein A-Sepharose beads. Bound DNA was extracted, PCR-amplified and used for the next round of CASTing. Three rounds of CASTing were performed. The amplified DNA fragments that interacted with Zbed4 were cloned and sequenced. <b>A</b>. Clones carrying poly-G tracts (bolded) and GC-box core sequences (underlined). <b>B</b>. Clones only carrying GC-box core sequences.</p

Determination of the minimal poly-G tract required for interaction with Zbed4 and of the affinity of Zbed4 for two different GC-box consensus sequences.

<p>Zbed4 (20 µg) was incubated with each of different 20-mer ssDNA primers containing G-tracts flanked by a different number of poly-As (0.5 nM), and with each of two GC-box consensus sequences for 45 min. The 20 µl reaction mixtures were loaded on a 1% agarose gel and EMSA was carried out as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035317#pone-0035317-g005" target="_blank">Figure 5</a>. A single primer (0.5 nM) and Zbed4 (20 µg) were used as controls. 1 kb DNA ladder was used as a standard for nucleic acid size. A. Agarose gel stained with SYBR Gold. B. The same gel stained with Coomassie R-250. As seen, the affinity of Zbed4 binds to for the oligonucleotides increases with the increasing number of Gs in the tracks. that contain at least 5 Gs and Quantification of the Zbed4-oligonucleotide complex bands showed that those with 5–11 Gs, 12–16 Gs and 17–18 Gs have similar density values: 105,415±15,217; 167,810±13,671; and 214,727±15,861, respectively. The complex with 19Gs has the highest value: 360,835. In addition, Zbed4 binds only to the GC-box, GGGGCGGGGC, indicating that the neighboring nucleotides of the core sequence are critical.</p