ADNP co-immunoprecipitates with splicing factors.

<p>A] Tau-Tg mice western blot analyses. Western blot analyses for Brm and ADNP were performed on 1- and 3-month-old-mice (3 or 4 replicates/age group, n = 5). One blot is shown, using actin as a control protein (results from 3–4 mice/experimental group are shown). B] Brm. Immunoprecipitation, gel electrophoresis (8% polyacrylamide) and western blot analysis was carried out as described in the method section. Increasing concentrations of nuclear protein extracts from the thalamus and subthalamic nucleus of 11-month-old C57BL/129 male mice were subjected to immunoprecipitation with Brm antibody (Sigma), 8% polyacrylamide gel electrophoresis and immunoblotting with either ADNP (BD) (lanes 1–3) or Brm antibody (4–6). The immunoprecipitates included the following gel lanes. Lane 1 & 4: 100 µg protein, 1 µg Brm antibody; lane 2 & 5: 200 µg protein, 1 µg Brm antibody; lane 3 & 6: 300 µg protein, 2 µg Brm antibody. C] PSF. As PSF apears in two molecular weights ∼100 KD and ∼49 KD (a proteolytic cleavage product <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087383#pone.0087383-Lee1" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087383#pone.0087383-ShavTal1" target="_blank">[34]</a>, we opted to use an immunoprecipitation kit (Pierce) that separates most antibodies used from the antigen that is then subjected to futher protein separation and immunodetection. Mouse cerebellar nuclear-enriched extracts (500 µg) from 8-month-old C57BL/129 male mice were subjected to immunoprecipitation with either 10 µg monoclonal PSF antibody (Sigma), (lanes 1) or 10 µg ADNP antibody (Bethyl, lane 2), followed by western blot analysis (10% polyacrylamide gel) with PSF antibody (1:2000 dilution; Sigma). As a control (lane 3), 500 µg of nuclear-enriched fraction from the thalamus and subthalamic nuclei were immunoprecipitated with 10 µg PSF antibody and subjected to ADNP western blot analysis (1:500 dilution; Bethyl, lane 3). D] Negative controls. Mouse nuclear protein extracts from the thalamus and subthalamic nucleus of 11-month-old C57BL/129 male mice (20 µg) were subjected only to western blot analysis to verify antibody binding (lane 1), the same nuclear-enriched extracts (500 µg) were further subjected to immunoprecipitation in absence of antibody (lane 2), for ADNP and Brm, western analysis was performed as in Fig. 5B PSF antibody (1:2000 dilution; Sigma) and ADNP antibody (1:500 dilution; Bethyl, lane 2). Mouse cerebellar nuclear-enriched extracts, (20 µg, lane 1, and 500 µg, lane 2) were further subjected to immunoprecipitation in absence of antibody, followed by western analysis with Brm antibody (1:1000 dilution; Sigma, as in Fig. 5C). Gel electrophoresis and extracts were as in 5B and C). E, F] Comparison between ADNP+/+ and ADNP+/- mice for Brm and PSF, ADNP interactions, respectively. Immunoprecipitation experiments were carried out as described in the materials and methods section. Western blot analysis was performed on the following samples: 1,5 Positive control (20 µg cortical lysate). 2,6 Negative control (IP without ADNP antibody). 3,7 First elution from the antibody affinity binding and specific antibody detection (as marked on the figure). 4,8 Second elution from the antibody affinity binding and specific antibody detection (as marked). 8% polyacrylamide gels were used for ADNP and Brm and 10% polyacrylamide was used for PSF. G] A suggested model of interaction.</p

Doxycycline treatment which shuts down transgene expression normalizes ADNP and tau 3R expression, while ADNP deficiency may regulate tau isotype expression.

<p>A] ADNP expression with doxycycline: Western blot analysis is depicted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087383#pone-0087383-g002" target="_blank">Fig. 2A</a>, on nuclear extracts with ADNP antibodies. When the ADNP/actin ratios were calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087383#pone-0087383-g001" target="_blank">Fig. 1</a>, a significant reduction of ADNP/actin ratio was observed following doxycycline treatment, correlating ADNP expression to tau transgene expression (*<i>p</i><0.05). Five mice were used per an experimental group; each gel lane includes one mouse extract with 3-4 mice representing an experimental group. B] ADNP mRNA quantification. The ADNP mRNA amount in each sample (n = 5/experimental group) was calculated using the correlated HPRT mRNA amount of the same sample. A comparison of Tg and non-Tg as well as Dox+ treatment is shown as for the proteins (panel A, above), (* <i>p</i><0.05). C] Trend toward deregulation of tau expression in the cortex of 2-month-old ADNP+/− mice. The expression of mouse tau 3R and mouse tau 4R (4–5 female mice, cerebral cortex/experimental group) was compared by quantitative real time PCR and results are depicted on the graph, comparing 2-month-old to 9-month-old. There was a significant difference (P<0.05, one tailed, Student's t-test) in tau 4R expression in the 2-month-old ADNP+/- mice compared to ADNP+/+ mice. Data is presented as fold-change (2^{−Δ<i>C</i>}_T), <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087383#pone.0087383-Schmittgen1" target="_blank">[55]</a>.</p

ADNP expression in the cerebellum is not subject to regulation by transgene expression in other brain areas.

<p>A] Western blot analysis. Expression of nuclear ADNP in the cerebellum of Tau-Tg mice (1) and non-Tg mice (2) is shown by gel electrophoresis and western blot analysis coupled to quantitative densitometry. Results show an increase in ADNP expression at 3 months of age in both transgenic and non-transgenic mice, followed by a decrease at 5.5 and 9 months of age (each experimental group included 4 mice, 3–4 representatives are shown). B] ADNP quantification in the cerebellum. The ADNP amount in each band was calculated as its percentage from the total amount of all bands, and was then divided by the correlated actin amount of the same sample. ADNP/actin amounts of each group were averaged [Tg: 1-month-old 0.43+0.078, 3-month- old 1.794+0.228, 5.5-month-old 1.009+0.08, 9-month-old 0.998+0.195. Non-Tg: 1-month-old 0.532+0.125, 3-month-old 1.798+0.264, 5.5-month-old 0.903+0.196, 9-month-old 0.406+0.166]. Four mice were used for each experimental point and gel electrophoresis was repeated twice, average results are shown. Statistical analysis is shown in the text.</p

Age-dependent changes in the expression of ADNP: Differences between Tau-Tg and control mice.

<p>A] Tau-Tg mice western blot analyses. Western blot analyses for ADNP were performed on 3-10.5-month-old-mice (3 or 4 replicates/age group, with 4–5 mice/group). One blot is shown, including extracts from 3-10.5-month-old-mice with actin as a control protein (each lane represents 1 mouse and 4 mice are shown per group). B] Age-dependent changes in the expression of ADNP at the protein level in the cerebral cortex: Differences between Tau-Tg and control mice. The ADNP/actin ratio in each immunoreactive band (in A) was calculated as the ADNP percentage from the total amount of all bands, and then divided by the correlating actin amount of the same sample. ADNP/actin ratios for each group were averaged (n = 5/group, average of 3–4 gel replicates). Quantitative densitometry is shown for four ages: 3, 5.5, 9 and 10.5 months (black bars, Tau-Tg; white bars, littermates not expressing the 4R mutated tau as outlined in the method section, *** <i>p</i><0.001, Tau-Tg vs. control mice, see results section). The 10.5-month point shows only the Tau-Tg ADNP expression. Additional experiments <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087383#pone.0087383-Gozes5" target="_blank">[54]</a> compared Tau-Tg and controls revealing no differences at the actin level (densitometry results of 8.4±3.4, vs. 8.3±2.6). In contrast, ADNP levels were significantly decreased in the Tau-Tg 5.5±0.9, vs. controls 11.2±1.35, <i>p</i><0.01 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087383#pone.0087383-Gozes5" target="_blank">[54]</a>. The insert is showing a western blot comparing ADNP expression in Tau-Tg (n = 4) and controls (n = 3), at the age of 3 months, each lane represents 1 mouse. C] Age-dependent changes in the expression of ADNP mRNA in the cerebral cortex: Differences between Tau-Tg and control mice. The ADNP mRNA amount in each sample was calculated using the correlated HPRT mRNA amount of the same sample. ADNP mRNA amounts for each group were averaged. [Tg: 1-month-old 9.422+<u>0.92</u>, 3-month-old 2.788±0.407, 5.5-month-old 1.242±0.314, 9-month-old 1.402±0.642. Non-Tg: 1-month-old 2.<u>78+0.28, 3</u>-month-old 1±0.357, 5.5-month-old 1.222±0.535, 9-month-old 2.18±0.27, n = 5/age group]. RQ  =  relative quantity (<a href="http://de-de.invitrogen.com/etc/medialib/en/filelibrary/Nucleic-Acid-Amplification-Expression-Profiling/PDFs.Par.83765.File.dat/relative-quant-ct.pdf" target="_blank">http://de-de.invitrogen.com/etc/medialib/en/filelibrary/Nucleic-Acid-Amplification-Expression-Profiling/PDFs.Par.83765.File.dat/relative-quant-ct.pdf</a>), (*** <i>p</i><0.01).</p