Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Transfer, analysis and reversion of the fibrous dysplasia cellular phenotype in human skeletal progenitors

doi: 10.1359/jbmr.091036

Figure Lengend Snippet: Table III

Article Snippet: PDE10A , hCG34059 , Hs01098924_m1.

Techniques:

Journal: Environment international

Article Title: Exposure to dithiocarbamate fungicide maneb in vitro and in vivo: Neuronal apoptosis and underlying mechanisms.

doi: 10.1016/j.envint.2022.107696

Figure Lengend Snippet: Fig. 7. Schematic diagram of the mechanisms underlying maneb-induced apoptosis. The mitochondrial apoptosis pathway (Bcl-2, Bax, cytochrome c, caspase-3) and the PKA/CREB pathway (PKA, PDE10A, CREB, p-CREB) are involved in maneb-induced apoptosis. In addition, maneb induces mitochon drial dysfunction and ROS accumulation, contributing to the neurotoxicity of maneb.

Article Snippet: The following antibodies and reagents were used: Bax (Proteintech, 50599–2-Ig), Bcl-2 (Cell Signaling Technology, 3498S), cytochrome c (Abcam, ab133504), caspase-3 (Proteintech, 19677-1-AP), GAPDH (Proteintech, 60004-1- Ig), PKA (Proteintech, 55382-1-AP), phosphodiesterase 10A (PDE10A) (Proteintech, 18078-1-AP), CREB (Proteintech, 12208-1-AP), p-CREB (ABclonal, AP0333), COX IV (Abcam, ab16056), DAPI (Biofroxx, EZ3412B205), MAP-2 (ThermoFisher, SF254293), HRP-conjugated antirabbit IgG (BIO-RAD, 170-6515), HRP-conjugated anti-mouse IgG (BIORAD, 170-6516), Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen, A-11001), Cell Counting Kit-8 Viability Assay (CCK-8) (Solarbio, CK04), Apoptosis and Necrosis Assay Kit (Beyotime Biotechnology, C1056), TUNEL BrightRed Apoptosis Detection Kit (Vazyme, A113), Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology, C1062), Reactive Oxygen Species Assay Kit (Nanjing Jiancheng Bioengineering Institute, E004-1-1), N-acetylcysteine (NAC) (SigmaAldrich, A7250), JC-10 (Solarbio, CA1310), Dibutyryl-cAMP (dbcAMP) (Selleck, S7858).

Techniques:

Journal: PLoS ONE

Article Title: Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa

doi: 10.1371/journal.pone.0161035

Figure Lengend Snippet: Oligonucleotide primers used for PCR amplification.

Article Snippet: Monoclonal anti-PDE10A antibody (5 μg; clone 1C9, Origene) was added to the pre-cleared fraction and incubated for 2 h. Protein G-coated sepharose beads were added and the suspension was incubated for another 2 h with constant rotation.

Techniques: Amplification

Journal: PLoS ONE

Article Title: Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa

doi: 10.1371/journal.pone.0161035

Figure Lengend Snippet: RNA isolated from bovine testis, testicular tetraploid (spermatocytes) and haploid (spermatids) germ cells was reverse transcribed and the transcripts encoding PDE10A variant X3 and X5 were amplified by PCR using specific primers. Amplification products obtained with spermatid RNA from 2 different bulls are shown. Similar results were obtained with RNA isolated from the testis, spermatocytes and spermatids from another bull. The expected size of the amplicons is 2367 bp and 2349 bp for variants X3 and X5, respectively. The position of molecular weight standards (bp) is shown on the left.

Article Snippet: Monoclonal anti-PDE10A antibody (5 μg; clone 1C9, Origene) was added to the pre-cleared fraction and incubated for 2 h. Protein G-coated sepharose beads were added and the suspension was incubated for another 2 h with constant rotation.

Techniques: Isolation, Reverse Transcription, Variant Assay, Amplification, Molecular Weight

Journal: PLoS ONE

Article Title: Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa

doi: 10.1371/journal.pone.0161035

Figure Lengend Snippet: Bull testicular sections were processed for immunohistochemistry as detailed in Materials & Methods using a monoclonal anti-PDE10A antibody (3G9; Panel A, C, D, E, and F) or non-immune commercial mouse IgG (Panel B). Positive signal (arrows in all Panels) appears as a brownish precipitate. Panels A and B show 400X magnification and Panels C—F show 1000X magnification. Sc stands for primary spermatocytes, Sd stands for round spermatids, and Es stands for elongated spermatids.

Article Snippet: Monoclonal anti-PDE10A antibody (5 μg; clone 1C9, Origene) was added to the pre-cleared fraction and incubated for 2 h. Protein G-coated sepharose beads were added and the suspension was incubated for another 2 h with constant rotation.

Techniques: Immunohistochemistry

Journal: PLoS ONE

Article Title: Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa

doi: 10.1371/journal.pone.0161035

Figure Lengend Snippet: Washed spermatozoa were subjected to nitrogen cavitation and the homogenate was centrifuged at low speed and then at high speed to separate raw membranes and cytosolic fractions as described in Materials & Methods. Equal amount of proteins from total cavitated sperm and from the membrane and cytosolic fractions were subjected to electrophoresis, transferred onto a PVDF membrane and probed with a PDE10A monoclonal antibody (clone 1C9). An anti-mouse IgG secondary antibody conjugated to horseradish peroxidase was then used and the positive signal was revealed with ChemiDoc MP Imaging system. Molecular weight standards (kDa) are indicated on the left.

Article Snippet: Monoclonal anti-PDE10A antibody (5 μg; clone 1C9, Origene) was added to the pre-cleared fraction and incubated for 2 h. Protein G-coated sepharose beads were added and the suspension was incubated for another 2 h with constant rotation.

Techniques: Membrane, Electrophoresis, Imaging, Molecular Weight

Journal: PLoS ONE

Article Title: Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa

doi: 10.1371/journal.pone.0161035

Figure Lengend Snippet: Washed spermatozoa were fixed with formaldehyde and permeabilized with Triton X-100. They were next incubated overnight with a monoclonal anti-PDE10A antibody (clone 3G9; A-F) or a commercial non-immune mouse IgG at the same concentration (G-I). Panels A, D & G show the positive immunofluorescent signal obtained using a DyLight 594-conjugated secondary antibody; Panels E & H show the presence/absence of the acrosome (PNA-FITC labeling) on spermatozoa from the same field of D & G, respectively. Panels B, F & I are Phase contrast image of the same respective field (A, D & G). Panel C shows the superposition of Panels A & B. Magnification is 1000X. Note that PDE10A is detected in the acrosomal region of acrosome intact (AI) but not of acrosome reacted (AR) sperm (D-F). Some non-specific staining is observed in the midpiece of both AI and AR sperm incubated with non-immune mouse IgG (G-I). Identical PDE10A localization is obtained when the immunodetection experiment is performed in the absence (A) or presence (D) of PNA-FITC, or using a FITC-conjugated secondary antibody (not shown).

Article Snippet: Monoclonal anti-PDE10A antibody (5 μg; clone 1C9, Origene) was added to the pre-cleared fraction and incubated for 2 h. Protein G-coated sepharose beads were added and the suspension was incubated for another 2 h with constant rotation.

Techniques: Incubation, Concentration Assay, Labeling, Staining, Immunodetection

Journal: PLoS ONE

Article Title: Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa

doi: 10.1371/journal.pone.0161035

Figure Lengend Snippet: Freshly ejaculated bull spermatozoa were washed, fixed with acrolein and pelleted in agarose. Sections of 50 μm were obtained and processed for immunodetection as described in Materials & Methods using monoclonal anti PDE10A antibody (A & B) or non-immune commercial mouse IgG (C & D). Sections were then post-fixed, embedded, and ultrathin sections were prepared and observed by transmission electron microscopy. Note that there is some non-specific signal in the midpiece region that is observed with both the PDE10A antibody and the non-immune IgG (arrowheads in A, B & C). Photographs were taken with magnification of 6800 X (A & C) and 9300 X (B & C). Acrosomal matrix (acr), plasma membrane (pm), and outer acrosomal membrane (oam; lining the acrosomal matrix) are indicated in the sperm head; mitochondria (mit), microtubules (mit) and outer dense fibers (odf) are indicated in the sperm midpiece. In all the panels, the bar scale represents 1 μm.

Article Snippet: Monoclonal anti-PDE10A antibody (5 μg; clone 1C9, Origene) was added to the pre-cleared fraction and incubated for 2 h. Protein G-coated sepharose beads were added and the suspension was incubated for another 2 h with constant rotation.

Techniques: Immunodetection, Transmission Assay, Electron Microscopy, Clinical Proteomics, Membrane

Journal: PLoS ONE

Article Title: Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa

doi: 10.1371/journal.pone.0161035

Figure Lengend Snippet: A) Ejaculated bull spermatozoa were processed for Immunoprecipitation as described in Materials & Methods using a PDE10A monoclonal antibody and protein G-coupled sepharose. The immune-complex on the beads was next solubilized with sample buffer, subjected to electrophoresis and transferred onto PVDF membrane. Proteins were next probed with a monoclonal anti-PDE10A antibody and revealed with a secondary antibody conjugated to horseradish peroxidase, enhanced chemiluminescence, and film exposure. Molecular weight standards (kDa) are indicated on the left. B) Coverage of the PDE10A isoform X4 amino acid sequence by the trypsin generated peptides identified by LC-MS/MS. Sequence sections highlighted in yellow matched the peptides identified. Amino acids highlighted in green show variable post-translational modifications (Oxidation of M, Deamination of Q or N).

Article Snippet: Monoclonal anti-PDE10A antibody (5 μg; clone 1C9, Origene) was added to the pre-cleared fraction and incubated for 2 h. Protein G-coated sepharose beads were added and the suspension was incubated for another 2 h with constant rotation.

Techniques: Immunoprecipitation, Electrophoresis, Membrane, Molecular Weight, Sequencing, Generated, Liquid Chromatography with Mass Spectroscopy

Journal: PLoS ONE

Article Title: Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa

doi: 10.1371/journal.pone.0161035

Figure Lengend Snippet: Intron-exon distribution deduced from the sequences obtained in our 5'-RLM (upper part) and 3'-RACE (lower part) experiments is presented. The numbers in the boxes refer to the number of nucleotides in the putative exons. The length of the introns is also indicated between the boxes. The position of the start of the ORFs in the variant X3 (isoform X2; MSHEPAEGGLDACDAS ), the variant X5 (isoform X4; MTFCGMARWY ), as well as the initial Methionine residue deduced from the different clones obtained in our 5’-RLM experiments that were similar to the variant X5 although with deletions in the 5’-UTR ( MQGVVYELNS ), are indicated. This ‘alternate’ start codon lies in the portion of the protein that is shared by all predicted PDE10A isoforms.

Article Snippet: Monoclonal anti-PDE10A antibody (5 μg; clone 1C9, Origene) was added to the pre-cleared fraction and incubated for 2 h. Protein G-coated sepharose beads were added and the suspension was incubated for another 2 h with constant rotation.

Techniques: Variant Assay, Residue, Clone Assay

Journal: Cardiovascular Research

Article Title: Role of PDE10A in vascular smooth muscle cell hyperplasia and pathological vascular remodelling

doi: 10.1093/cvr/cvab304

Figure Lengend Snippet: PDE10A expression level is increased in growing SMCs in vitro and SMC-like cells in intimal lesions in vivo. (A) RT-qPCR results showing relative PDE10A mRNA levels in rat SMCs stimulated with FBS for different amounts of time. Cells were starved for 2 days before being treated with 10% FBS. n = 3 for each group. A one-sample t-test was performed to determine the significance of the fold change. (B) RT-qPCR results showing PDGF-induced PDE10A expression in rat SMCs. Cells were serum-deprived for 2 days and then treated with 10 ng/mL PDGF-BB for 3 h. A one-sample t-test was conducted. n = 3 for each group. (C) Immunoblots showing PDE10A and GAPDH levels of rat SMCs with FBS stimulation. Cells were starved for 2 days and then incubated with 10% FBS for 6 h. (D) Quantitative results of (C). n = 3 for each group. The signal of PDE10A bands was first normalized to the input and then normalized to the group without FBS treatment. A one-sample t-test was performed. n = 3 for each group. (E) Representative images of the proximity ligation assay on PDE10A expression in cross-sections of wire-injured mouse femoral arteries. The left femoral arteries were injured and the right femoral arteries served as sham controls. Femoral arteries were harvested 4 weeks after the surgery. Yellow dash lines indicate the internal and external elastic laminae. A, adventitia; I, intima; M, media. Similar observations were obtained from tissue samples from at least three different mice. Scale bar = 20μm. All data are shown as mean ± SEM.

Article Snippet: Two primary PDE10A antibodies (Santa Cruz, sc-515023 and Novus Biologicals, NB300-645) raised in mice and rabbits that recognize different epitopes of the PDE10A protein were used.

Techniques: Expressing, In Vitro, In Vivo, Quantitative RT-PCR, Western Blot, Incubation, Proximity Ligation Assay

Journal: Cardiovascular Research

Article Title: Role of PDE10A in vascular smooth muscle cell hyperplasia and pathological vascular remodelling

doi: 10.1093/cvr/cvab304

Figure Lengend Snippet: The effects of PDE10A inhibition or knocking down on SMC proliferation or death. (A) Cell numbers of rat SMCs after being starved for 2 days and then stimulated by 2% FBS in the presence of different doses of MP-10. A Welch’s ANOVA with Dunnett’s T3 multiple comparisons test was performed. n = 3 for each group. (B) Cell numbers of rat SMCs with PDE10A knockdown by two different pairs of shRNAs, after being starved and stimulated with FBS. Cells were treated with lentivirus-mediated shRNAs 3 days before FBS stimulation. A Welch’s ANOVA with Dunnett’s T3 multiple comparisons test was carried out. n = 6 for each group. (C) RT-qPCR results showing the efficiency of PDE10A knockdown. The expression level of PDE10A was normalized to the control shRNA group. One-sample t-tests were performed. n = 3 for each group. (D) Representative immunofluorescent images of BrdU positive rat SMCs treated with vehicle or MP-10 (5 μM). Cells were starved for 2 days and then stimulated by 2% FBS. (E) Quantitative data showing relative ratios of BrdU positive SMCs. The ratios were normalized to the group without FBS stimulation. An unpaired Student’s t-test with Welch’s correction was conducted to compare the FBS-stimulated proliferation without and with MP-10. n = 4 for each group. (F) Relative ratios of BrdU positive SMCs after PDE10A silencing by shRNAs. A one-way ANOVA with Bonferroni’s multiple comparisons test was applied. n = 3 for each group. (G) Representative images of TUNEL staining of rat SMCs after 5 μM MP-10 treatment. Red signals indicate apoptotic cells. H2O2 was used as a positive control. (H) Quantitative data of the TUNEL staining in (G). The percentages of TUNEL positive cells against DAPI (total cells) were calculated. A Kruskal–Wallis test was performed. n = 3 for each group. Scale bar = 50μm. All data are shown as mean ± SEM. ns, not significant.

Article Snippet: Two primary PDE10A antibodies (Santa Cruz, sc-515023 and Novus Biologicals, NB300-645) raised in mice and rabbits that recognize different epitopes of the PDE10A protein were used.

Techniques: Inhibition, Knockdown, Quantitative RT-PCR, Expressing, Control, shRNA, TUNEL Assay, Staining, Positive Control

Journal: Cardiovascular Research

Article Title: Role of PDE10A in vascular smooth muscle cell hyperplasia and pathological vascular remodelling

doi: 10.1093/cvr/cvab304

Figure Lengend Snippet: Cell cycle arrest of SMCs induced by PDE10A inhibition or silencing. (A) Percentages of rat SMCs in different phases of a cell cycle after being treated with MP-10. Rat SMCs were starved for 2 days, followed by 2% FBS stimulation. The blue boxes indicate cells in the G1/G0 phase, the yellow boxes show cells in the S phase and the green colour is for cells in the G2/M phase. (B) Percentages of S-phase cells after being treated with MP-10 in addition to FBS. A Welch’s ANOVA with Dunnett’s T3 multiple comparisons test was performed. n = 3 for each group. (C) Percentages of S-phase cells after being stimulated by PDGF (10 ng/mL) and treated with MP-10. A Welch’s ANOVA with Dunnett’s T3 multiple comparisons test was conducted. n = 3 for each group. The percentages of cells in (D) all phases or in (E) S-phase alone after cells being knocked down of PDE10A and stimulated via 10 ng/mL PDGF. Cells were transduced by lentiviruses expressing different PDE10A shRNAs 3 days before PDGF stimulation. A Welch’s ANOVA with Dunnett’s T3 multiple comparisons test was implemented. n = 3 for each group. (F) Western blot showing the expression of cyclin D1 at different time points after SMCs were starved and then stimulated by 2% FBS with vehicle or 5 μM MP-10. The upper part shows the relative quantification results, while the lower part exhibits the representative images of immunoblot staining of cyclin D1, CDK4, CDK6, and α-tubulin. Unpaired Student’s t-tests were conducted on comparison within each time point. n = 3–4 for each group. All data are shown as mean ± SEM.

Article Snippet: Two primary PDE10A antibodies (Santa Cruz, sc-515023 and Novus Biologicals, NB300-645) raised in mice and rabbits that recognize different epitopes of the PDE10A protein were used.

Techniques: Inhibition, Expressing, Western Blot, Quantitative Proteomics, Staining, Comparison

Journal: Cardiovascular Research

Article Title: Role of PDE10A in vascular smooth muscle cell hyperplasia and pathological vascular remodelling

doi: 10.1093/cvr/cvab304

Figure Lengend Snippet: PDE10A modulated SMC proliferation involves cGMP signalling pathway. (A) cAMP ELISA results showing the change of cAMP concentration after rat SMCs were treated with 5 μM MP-10. An unpaired Student’s t-test was performed. n = 3 for each group. (B) cGMP concentration changes after MP-10 treatment revealed by cGMP ELISA. An unpaired Student’s t-test with Welch’s correction was conducted. n = 3 for each group. (C) Percentages of S-phase cells after being stimulated by 10 ng/mL PDGF and treated with 2.5 μM MP-10 and 10 μM PKI (a PKA inhibitor). Cells were starved for 2 days before PDGF stimulation. A Welch’s ANOVA with Dunnett’s T3 multiple comparison test was performed to determine the effects of MP-10 and PKI on SMC proliferation. n = 3 for each group. (D) Percentages of S-phase cells treated with 2.5 μM MP-10 and 5 μM DT-2 (a PKG inhibitor). A Welch’s ANOVA with Dunnett’s T3 multiple comparisons test was applied. n = 3–4 for each group. (E) RT-qPCR results showing the mRNA levels of PKG1α and PKG1β after knocking down PKG1α in rat SMCs. The expression levels were normalized to negative control siRNA groups. One-sample t-tests were performed to determine the knockdown efficiency. n = 3 for each group. (F) RT-qPCR results showing the levels of PKG1α and PKG1β after PKG1β silencing. One-sample t-tests were conducted. n = 3 for each group. (G) Percentages of S-phase cells treated with control or PKG1α siRNA and 2.5 μM MP-10. A Welch’s ANOVA with Dunnett’s T3 multiple comparisons test was applied. n = 4 for each group. (H) Percentages of S-phase cells treated with PKG1β siRNA and 2.5 μM MP-10. A Welch’s ANOVA with Dunnett’s T3 multiple comparisons test was applied. n = 3 for each group. All data are shown as mean ± SEM. ns, not significant.

Article Snippet: Two primary PDE10A antibodies (Santa Cruz, sc-515023 and Novus Biologicals, NB300-645) raised in mice and rabbits that recognize different epitopes of the PDE10A protein were used.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Comparison, Quantitative RT-PCR, Expressing, Negative Control, Knockdown, Control

Journal: Cardiovascular Research

Article Title: Role of PDE10A in vascular smooth muscle cell hyperplasia and pathological vascular remodelling

doi: 10.1093/cvr/cvab304

Figure Lengend Snippet: PDE10A-modulated SMC proliferation is associated with CNP/NPR2 signalling pathway. (A) Relative mRNA levels of NPR1 and NPR2 in rat SMCs. An unpaired Student’s t-test with Welch’s correction was performed. n = 3 for each group. (B) RT-qPCR showing the level of NPR2 in SMCs treated with control or NPR2 siRNA. The expression level was normalized to the control siRNA-treated group. A one-sample t-test was performed to determine the knockdown efficiency. n = 3 for each group. (C) Percentages of S-phase cells treated with 2.5 μM MP-10, NPR2 siRNA, or both. A Welch’s ANOVA with Dunnett’s T3 multiple comparisons test was applied. n = 3 for each group. (D) RT-qPCR results showing the level of NPPC (encoding a precursor of CNP) in SMCs treated with control or NPRC siRNA. The mRNA level was normalized to the control siRNA-treated group. A one-sample t-test was performed to determine the knockdown efficiency. n = 3 for each group. (E) Percentages of S-phase cells treated with 2.5 μM MP-10, NPPC siRNA, or both. A Welch’s ANOVA with Dunnett’s T3 multiple comparisons test was applied. n = 3 for each group. (F) Percentages of rat SMCs in the S-phase after being treated with 2.5 μM MP-10, 1 μM CNP, or both. A Welch’s ANOVA with Dunnett’s T3 multiple comparisons test was performed. n = 3 for each group. (G and H) Confocal microscopic images (left panels) showing the signals of EGFP-tagged PDE10A (green) and mCherry-tagged NPR2 (red) overexpressed in 293A cells (G) or rat SMCs (H). Scale bar = 10μm (G) or 20 μm (H). The intensities of green and red signals across the cell were measured (right panels). (I) Immunoblot showing PDE10A and NPR2 from co-immunoprecipitation. Flag-tagged PDE10A and HA-tagged NPR2 were overexpressed in 293A cells. All data are shown as mean ± SEM. n = 3 for each group. ns, not significant.

Article Snippet: Two primary PDE10A antibodies (Santa Cruz, sc-515023 and Novus Biologicals, NB300-645) raised in mice and rabbits that recognize different epitopes of the PDE10A protein were used.

Techniques: Quantitative RT-PCR, Control, Expressing, Knockdown, Western Blot, Immunoprecipitation

Journal: Cardiovascular Research

Article Title: Role of PDE10A in vascular smooth muscle cell hyperplasia and pathological vascular remodelling

doi: 10.1093/cvr/cvab304

Figure Lengend Snippet: Neointimal formation in femoral arteries of PDE10A-WT and PDE10A-KO mice after wire injury. (A) Representative histological images of cross-sections of femoral arteries from male WT and KO mice. The right femoral arteries were undergone wire injury and the left femoral arteries served as sham controls. Elastin in the internal and external laminae was stained black. (B) Intimal area, (C) medial area, and (D) the intima/media ratio of femoral arteries of male WT and KO mice. Five sections, 300 µm apart, were measured and averaged for each animal. An unpaired Student’s t-test was used for comparing the intima area. Mann–Whitney tests were used for the medial area and the intima/media ratio. n = 19 mice for WT and n = 21 mice for KO. (E) Intimal area, (F) medial area, and (G) the intima/media ratio of femoral arteries of female WT and KO mice. An unpaired Student’s t-test was used for comparing the media area. Mann–Whitney tests were used for the intima area and the intima versus media ratio. n = 18 mice for WT and n = 15 mice for KO. Scale bar = 50μm. All data are shown as mean ± SEM.

Article Snippet: Two primary PDE10A antibodies (Santa Cruz, sc-515023 and Novus Biologicals, NB300-645) raised in mice and rabbits that recognize different epitopes of the PDE10A protein were used.

Techniques: Staining, MANN-WHITNEY

Journal: Neuropsychopharmacology

Article Title: TAK-063, a PDE10A Inhibitor with Balanced Activation of Direct and Indirect Pathways, Provides Potent Antipsychotic-Like Effects in Multiple Paradigms

doi: 10.1038/npp.2016.20

Figure Lengend Snippet: PDE10A Occupancy, Striatal cAMP Increase, and Suppression of MK-801-Induced Hyperactivity by TAK-063 and MP-10 in Mice and Rats

Article Snippet: Homozygous Pde10a -knockout mice and their wild-type littermates were purchased from Taconic Biosciences, Inc. (Hudson, NY), and backcrossed to C57BL/6J background.

Techniques: Inhibition

Journal: Neuropsychopharmacology

Article Title: TAK-063, a PDE10A Inhibitor with Balanced Activation of Direct and Indirect Pathways, Provides Potent Antipsychotic-Like Effects in Multiple Paradigms

doi: 10.1038/npp.2016.20

Figure Lengend Snippet: TAK-063, but not MP-10, produced significant and dose-dependent antipsychotic-like effects in METH-induced hyperactivity and PPI deficits. (a) Suppression of METH or MK-801-induced hyperactivity in rats. METH (0.5 mg/kg) or MK-801 (0.3 mg/kg) was administered to rats subcutaneously 1.5 or 0.5 h after oral administration of TAK-063 or MP-10, respectively. Accumulated activity counts during the 2 h after METH or MK-801 treatment were calculated and are indicated as mean+SEM (n=4–6). PDE10A occupancy at each dose used is shown below the dose. **P⩽0.05 vs control by Aspin–Welch test. #P⩽0.05 vs the vehicle+METH group by two-tailed Williams' test (Williams, 1971). §P⩽0.05 vs vehicle by two-tailed Shirley–Williams test (Shirley, 1977). (b) The effects of TAK-063 and MP-10 on PPI in C57BL/6J mice. Risperidone (RIS 1, 1 mg/kg) was used as a control. PDE10A occupancy at each doses used is shown below the dose. Data (%PPI to the 82-dB prepulse) are presented as mean+SEM. (n=10–13). §P⩽0.05 vs vehicle by two-tailed Shirley–Williams test. **P⩽0.01 vs vehicle by Aspin-Welch test. (c) The effect of TAK-063 and MP-10 on MK-801-induced PPI deficits in ICR mice. Risperidone (RIS 1, 1 mg/kg) was used as a control. PDE10A occupancy at each dose used is shown below the dose. Data (%PPI to the 82-dB prepulse) are presented as mean+SEM (n=8–16). **P⩽0.01 vs vehicle by Aspin–Welch test. #P⩽0.05 vs vehicle by two-tailed Williams' test. See also Supplementary Figures S2 and S3. METH, methamphetamine; ICR, imprinting control region; PPI, prepulse inhibition.

Article Snippet: Homozygous Pde10a -knockout mice and their wild-type littermates were purchased from Taconic Biosciences, Inc. (Hudson, NY), and backcrossed to C57BL/6J background.

Techniques: Produced, Activity Assay, Control, Two Tailed Test, Inhibition

Journal: Neuropsychopharmacology

Article Title: TAK-063, a PDE10A Inhibitor with Balanced Activation of Direct and Indirect Pathways, Provides Potent Antipsychotic-Like Effects in Multiple Paradigms

doi: 10.1038/npp.2016.20

Figure Lengend Snippet: Higher cyclic nucleotide concentrations in the direct pathway and higher sensitivity of TAK-063 for binding inhibition by cyclic nucleotides would lead to a partial PDE10A inhibition by TAK-063 in the direct pathway MSNs. (a) Representative images of cAMP and substance P staining in coronal sections of the rat striatum at 0.5 h after treatment with vehicle or MP-10 (30 mg/kg p.o.). The images were processed using NIS-Elements AR to adjust brightness and contrast. The arrowheads indicate representative cells double positive for anti-cAMP and anti-substance P staining. Scale bar, 10 μm. (b) Elevation of cAMP and cGMP levels in the rat striatum at 2 and 0.5 h after administration of TAK-063 and MP-10, respectively. PDE10A occupancy at each dose used is shown below the dose. Data are presented as mean+SEM (n=7). #P⩽0.05 vs the vehicle-treated group by two-tailed Williams' test. §P⩽0.05 vs the vehicle-treated group by two-tailed Shirley-Williams test. (c) Binding of TAK-063 and MP-10 to PDE10A in rat brain coronal sections in the presence or absence of cyclic nucleotides (cAMP and cGMP) at levels of 0.6 mM (0.3 mM each), 6 mM (3 mM each), and 60 mM (30 mM each). Data are presented as mean±SEM (n=4). The values in the graph represent binding levels relative to those in the absence of cyclic nucleotides. Data were analyzed by two-way ANOVA followed by Aspin–Welch test. **P⩽0.01. (d) The binding affinities of TAK-063 and MP-10 to PDE10A in rat coronal sections were determined by competitive binding assays using [3H]T-773. Data are presented as mean±SEM (n=3). ANOVA, analysis of variance; cAMP, cyclic AMP; cGMP, cyclic GMP; MSN, medium spiny neuron.

Article Snippet: Homozygous Pde10a -knockout mice and their wild-type littermates were purchased from Taconic Biosciences, Inc. (Hudson, NY), and backcrossed to C57BL/6J background.

Techniques: Binding Assay, Inhibition, Staining, Two Tailed Test

Journal: Neuropsychopharmacology

Article Title: TAK-063, a PDE10A Inhibitor with Balanced Activation of Direct and Indirect Pathways, Provides Potent Antipsychotic-Like Effects in Multiple Paradigms

doi: 10.1038/npp.2016.20

Figure Lengend Snippet: Compared with TAK-063, MP-10 induced greater levels of direct pathway activation and dopamine release that can cancel antipsychotic effect by D2 antagonism. (a) Effect of SKF82958 on the antipsychotic-like effect of haloperidol in METH-induced hyperactivity in rats. METH (0.5 mg/kg) was administered subcutaneously 1 and 0.5 h after administration of haloperidol (HAL, 0.3 mg/kg p.o.) and SKF82958 (SKF, 1, 3, and 10 mg/kg i.p.), respectively. Accumulated activity counts during the 2 h after METH treatment were calculated and are indicated as mean+SEM (n=5). **P⩽0.01 between the control group and METH-treated group by Aspin–Welch test. *P⩽0.05 between the METH-treated group and HAL+METH-treated group by Aspin–Welch test. #P⩽0.05 vs the HAL+vehicle-treated group by two-tailed Williams' test. (b) Three hours after administration of TAK-063 or MP-10, mRNA expression levels of substance P (SP, a marker of the direct pathway) and enkephalin (Enk, a marker of the indirect pathway) in the rat striatum were measured by PCR analysis. PDE10A occupancy at each dose used is shown below the dose. The values in the graph represent expression levels relative to that of the vehicle-treated group. Data are presented as mean+SEM (n=6). §P⩽0.05 vs the vehicle-treated group by two-tailed Williams' test (for SP expression). #P⩽0.05 vs the vehicle-treated group by two-tailed Williams' test (for Enk expression). (c) Ratio of substance P mRNA to enkephalin mRNA after PDE10A inhibition. PDE10A occupancy at each dose used is shown below the dose. The values in the graph represent expression ratio of substance P to enkephalin (SP/Enk) relative to that of the vehicle-treated group. Data are presented as mean+SEM (n=6). #P⩽0.05 vs the vehicle-treated group by two-tailed Williams' test. (d) Striatal dopamine release during continuous injection of SKF82958 (100 μM, 1 μl/min) into the rat striatum for 60 min. SKF82958 was continuously injected from time 0. Data are expressed as the percentage of baseline dopamine levels and are mean+SEM (n=6). **P⩽0.01 between the SKF82958-injected group and vehicle-injected group at 60 min after the start of injection by Aspin–Welch test. (e) Striatal dopamine release by TAK-063 or MP-10 was measured using microdialysis in rats. Drugs were administered at time 0. Data are presented as the percentage of baseline dopamine levels and are mean+SEM (n=5–6). RM-ANOVA was followed by Williams' test (for parametric data, P>0.05 by Bartlett's test) or Shirley–Williams test (for non-parametric data, P⩽0.05 by Bartlett's test). #P⩽0.05 vs the vehicle-treated group by two-tailed Williams' test at each time point. (f) Striatal dopamine release by TAK-063 and MP-10 was measured using microdialysis in Pde10a-knockout mice (KO) and their wild-type littermates (WT). Drugs were administered at time 0. Data are presented as the percentage of baseline dopamine levels and are mean+SEM (n=3–5). METH, methamphetamine; RM-ANOVA, repeated measures analysis of variance.

Article Snippet: Homozygous Pde10a -knockout mice and their wild-type littermates were purchased from Taconic Biosciences, Inc. (Hudson, NY), and backcrossed to C57BL/6J background.

Techniques: Activation Assay, Activity Assay, Control, Two Tailed Test, Expressing, Marker, Inhibition, Injection, Knock-Out

Journal: Neuropsychopharmacology

Article Title: TAK-063, a PDE10A Inhibitor with Balanced Activation of Direct and Indirect Pathways, Provides Potent Antipsychotic-Like Effects in Multiple Paradigms

doi: 10.1038/npp.2016.20

Figure Lengend Snippet: Off-rates of PDE10A inhibitors may characterize their pharmacological profiles via regulation of each MSN pathway. (a) Schematic illustration of the experimental procedure to evaluate off-rates of PDE10A inhibitors. The off-rates of TAK-063 and MP-10 from PDE10A in rat brain sections were obtained by measuring their PDE10A occupancy using [3H]T-773 as a tracer. (b) Comparison of the off-rates of TAK-063, MP-10, and compound 1 from PDE10A. Data are presented as mean+SEM (n=3). The PDE10A occupancies of TAK-063, MP-10, and compound 1 at 60-min incubation after saturation in rat brain sections were 23.09, 49.85, and 43.95%, respectively. (c) Suppression of METH-induced hyperactivity in rats. METH was administered 3 h after administration of compound 1. Accumulated activity counts during the 2 h after METH treatment were calculated. Data are presented as mean+SEM (n=5). *P⩽0.05 vs control by Aspin–Welch test. (d) The effects of compound 1 on PPI in C57BL/6J mice. Data are presented as mean+SEM (n=6–8). (e) Activation of MSNs by compound 1. mRNA levels of substance P (SP) and enkephalin (Enk) in the rat striatum were measured by quantitative PCR 3 h after administration of compound 1. Data are presented as mean+SEM (n=6). §P⩽0.05, #P⩽0.05 by two-tailed Williams' test. (f) Ratio of substance P mRNA to enkephalin mRNA after PDE10A inhibition. PDE10A occupancy at each dose is shown below the dose. The values in the graph represent ratio of substance P to enkephalin (SP/Enk) expression relative to that of the vehicle-treated group. Data are presented as mean+SEM (n=6). #P⩽0.05 vs the vehicle-treated group. (g) Striatal dopamine release by compound 1 was measured using microdialysis in rats. Data are presented as the percentage of baseline dopamine levels (n=6–7). RM-ANOVA was followed by Williams' test (for parametric data, P>0.05 by Bartlett's test) or Shirley-Williams test (for non-parametric data, P⩽0.05 by Bartlett's test). #P⩽0.05 vs the vehicle-treated group by two-tailed Williams' test at each time point. §P⩽0.05 vs the vehicle-treated group by two-tailed Shirley–Williams test at each time point. See also Supplementary Figure S5. METH, methamphetamine; MSN, medium spiny neuron; PPI, prepulse inhibition; RM-ANOVA, repeated measures analysis of variance.

Article Snippet: Homozygous Pde10a -knockout mice and their wild-type littermates were purchased from Taconic Biosciences, Inc. (Hudson, NY), and backcrossed to C57BL/6J background.

Techniques: Comparison, Incubation, Activity Assay, Control, Activation Assay, Real-time Polymerase Chain Reaction, Two Tailed Test, Inhibition, Expressing

Journal: Oncogene

Article Title: Phosphodiesterase 10A: a novel target for selective inhibition of colon tumor cell growth and β-catenin-dependent TCF transcriptional activity

doi: 10.1038/onc.2014.94

Figure Lengend Snippet: PDE10 expression is elevated in human colon cancer and intestinal tumors from Apc Min/+ mice. a, PDE10 protein levels in human normal colonocytes (NCM460) and colon tumor cell lines derived from adenocarcinomas (HCT116, HT29, Caco2, SW480) or adenomas (LT97) as determined by western blotting. The antibody recognizes a protein band with a MW of approximately 90kD, which corresponds to the predicted MW of PDE10. b, PDE10 mRNA expression in human normal colon and adenocarcinomas as determined by quantitative real-time PCR. c, Data for induction fold for each cancer stage in panel b are represented as box-and-whisker plots with minimum and maximum. The boxed area represents 50% of samples (from the 25 th to the 75 th percentile) and the band inside the box represents the median. d, expression of PDE10 in normal colonic mucosa ( i–ii ), adenocarcinoma and matched uninvolved mucosa distal to tumor ( iii–iv ), another adenocarcinoma ( v–vi ), adenoma ( vii ), and metastasis to liver capsule of colorectal cancer ( viii ) as determined by immunohistochemistry. Tissues were labeled either with a PDE10-specific antibody from Novus ( i to vi ) or a monoclonal PDE10 antibody from GeneTex ( vii and viii ), while controls with no antibody showed no labeling. Red arrows show the border of a colon tumor adjacent to uninvolved colorectal glands (blue arrows). The area within the blue squares is enlarged and presented to the right of image. Green arrows indicate the localization of PDE10 in perinuclear, nuclear, cytoplasmic and cell memebrane regions of the cell. The final magnification of i–iv and vii is 100×. The final magnification of v and viii is 200×. The final magnification of vi is 630×. e, PDE10 mRNA levels were increased in intestinal tumors from Apc Min/+ mice compared with normal intestinal mucosa from wild-type mice. f, PDE10 mRNA expression in paired normal intestine and tumors from Apc Min/+ mice. g , PDE10 mRNA levels in normal intestinal mucosa from Apc Min/+ mice and wild-type mice. All data in panel e–g are represented as mean±SEM of three independent experiments. *p<0.05, **p<0.01.

Article Snippet: NCM460 and LT97 cells were transiently transfected with a human full-length PDE10 cDNA (Origene, Rockville, MD, USA) using X-tremeGENE DNA transfection reagent (Roche, Indianapolis, IN, USA) for 72 hours.

Techniques: Expressing, Derivative Assay, Western Blot, Real-time Polymerase Chain Reaction, Whisker Assay, Immunohistochemistry, Labeling

Journal: Oncogene

Article Title: Phosphodiesterase 10A: a novel target for selective inhibition of colon tumor cell growth and β-catenin-dependent TCF transcriptional activity

doi: 10.1038/onc.2014.94

Figure Lengend Snippet: PDE10 inhibitors selectively inhibited colon tumor cell growth. a, chemical structures of papaverine ( i ), PQ-10 ( ii ) and Pf-2545920 ( iii ). b–d , dose-dependent cell growth-inhibitory activity of PDE10 inhibitors papaverine ( b ), PQ-10 ( c ), and Pf-2545920 ( d ) as measured by luciferase-based ATP assay after 72 hours of treatment. e, apoptosis induction of HT29 cells by Pf-2545920 after 24 hours of treatment. f, inhibition of proliferation by Pf-2545920 treatment after 24 hours in HT29 colon tumor cells. g , induction of G1 cell cycle arrest in HT29 cells after 48 hours of treatment with Pf-2545920. All data are represented as mean±SEM of three independent experiments. *p<0.05, **p<0.01.

Article Snippet: NCM460 and LT97 cells were transiently transfected with a human full-length PDE10 cDNA (Origene, Rockville, MD, USA) using X-tremeGENE DNA transfection reagent (Roche, Indianapolis, IN, USA) for 72 hours.

Techniques: Activity Assay, Luciferase, ATP Assay, Inhibition

Journal: Oncogene

Article Title: Phosphodiesterase 10A: a novel target for selective inhibition of colon tumor cell growth and β-catenin-dependent TCF transcriptional activity

doi: 10.1038/onc.2014.94

Figure Lengend Snippet: Knockdown of PDE10 exhibits potent anticancer activity. a, siRNA-mediated knockdown of PDE10 expression as determined by western blotting. b, PDE10 knockdown by siRNA selectively inhibited tumor cell growth. c, tumor cell specific induction of apoptosis by PDE10 siRNA. d, selective inhibition of tumor cell proliferation by PDE10 siRNA. e, siRNA-mediated PDE10 knockdown arrested HT29 colon tumor cells in the G 1 phase of the cell cycle. f, Stable PDE10 knockdown leads to a decrease of anchorage-independent colony formation in HT29 cells. Cells were grown in soft agar for 21 days then stained and counted. Images from a representative experiment are shown on the left, and statistical analysis is shown on the right. All data are represented as mean±SEM of three independent experiments. *p<0.05, **p<0.01.

Article Snippet: NCM460 and LT97 cells were transiently transfected with a human full-length PDE10 cDNA (Origene, Rockville, MD, USA) using X-tremeGENE DNA transfection reagent (Roche, Indianapolis, IN, USA) for 72 hours.

Techniques: Knockdown, Activity Assay, Expressing, Western Blot, Inhibition, Staining

Journal: Oncogene

Article Title: Phosphodiesterase 10A: a novel target for selective inhibition of colon tumor cell growth and β-catenin-dependent TCF transcriptional activity

doi: 10.1038/onc.2014.94

Figure Lengend Snippet: Knockdown of PDE10 by siRNA activates the cGMP/PKG pathway and inhibits Wnt/β-catenin signaling. a, selective suppression of cGMP hydrolysis by PDE10 siRNA in HCT116 and HT29 colon tumor cells, but not NCM460 colonocytes. b, no effect of PDE10 siRNA on cAMP hydrolysis in HCT116, HT29 and NCM460 cells. c, siRNA-mediated PDE10 knockdown activated PKG, but not PKA, as determined by increased phosphorylation of VASP at the serine 239 residue, but not the serine 157 residue, in HCT116 colon tumor cells. d, PDE10 siRNA suppressed the expression of β-catenin and survivin in HCT116 colon tumor cells. The numbers indicated below the blots (panel c and d ) derive from band quantification normalized to GAPDH and are expressed as ratio of scrambled control siRNA treatment. e, PDE10 knockdown by siRNA inhibited β-catenin-dependent TCF transcriptional activity. f, PDE10 knockdown decreased β-catenin mRNA levels in colon tumor cells at 72 hours post-transfection. β-catenin mRNA expression was normalized to GAPDH expression. g, suppression of CTNNB1 (β-catenin gene) transcriptional activity by PDE10 siRNA after 72 hours transfection in HCT116 and HT29 colon tumor cells. All data are represented as mean±SEM of three independent experiments. **p<0.01.

Article Snippet: NCM460 and LT97 cells were transiently transfected with a human full-length PDE10 cDNA (Origene, Rockville, MD, USA) using X-tremeGENE DNA transfection reagent (Roche, Indianapolis, IN, USA) for 72 hours.

Techniques: Knockdown, Phospho-proteomics, Residue, Expressing, Control, Activity Assay, Transfection

Journal: Oncogene

Article Title: Phosphodiesterase 10A: a novel target for selective inhibition of colon tumor cell growth and β-catenin-dependent TCF transcriptional activity

doi: 10.1038/onc.2014.94

Figure Lengend Snippet: Overexpression of PDE10 promotes colon cell growth. a, confirmation of ectopic expression of PDE10 in colonocytes (NCM460) and adenoma cells (LT97) by western blotting. b, ectopic expression of PDE10 promoted growth of colonocytes and adenoma cells after 72 hours transfection of PDE10-expressing vector compared with cells transfected with control vector. All data are represented as mean±SEM of three independent experiments. *p<0.05. c, PDE10 overexpression activated β-catenin signaling pathway as shown by increased expression of cyclin D1 and survivin, two genes that are regulated by β-catenin signaling, in PDE10-experssing vector transfected cells. d, schematic hypothesis for the mechanism underlying the antitumor activity of PDE10 inhibition in colon tumor cells. Inhibition of PDE10 elevates intracellular cGMP levels and activates PKG to suppress the expression of β-catenin and inhibit TCF transcription of target genes (e.g. cyclin D and survivin).

Article Snippet: NCM460 and LT97 cells were transiently transfected with a human full-length PDE10 cDNA (Origene, Rockville, MD, USA) using X-tremeGENE DNA transfection reagent (Roche, Indianapolis, IN, USA) for 72 hours.

Techniques: Over Expression, Expressing, Western Blot, Transfection, Plasmid Preparation, Control, Activity Assay, Inhibition

Journal: PLoS ONE

Article Title: HPRT-Deficiency Dysregulates cAMP-PKA Signaling and Phosphodiesterase 10A Expression: Mechanistic Insight and Potential Target for Lesch-Nyhan Disease?

doi: 10.1371/journal.pone.0063333

Figure Lengend Snippet: ( A&B ) Immuno-blot and quantification analysis for various PDEs that can affect cAMP/PKA signaling. Data show that the expression of PDE1C, PDE4B, and PDE7B are similar between control (parent) and HPRT-deficient (mutant) MN9D cell lines. Expression of PDE10A protein is significantly increased in HPRT-deficient cells. Error bars represent mean ± SEM of duplicate measurements of two independent experiments (n = 4). The asterisk (*) represents the statistical significant between the control (open bars) and HPRT-deficient (closed bars) MND9 cells (p<0.05 t-test).

Article Snippet: 2×10 5 HPRT-deficient MN9D cells in 6 well-plate were exposed to 80 pmole of control (scramble siRNA) or PDE10A siRNA (directed to mouse PDE10A) from Santa cruz biotechnology and transfected according to the established transfection protocol ( http://datasheets.scbt.com/siRNA_protocol.pdf ).

Techniques: Expressing, Control, Mutagenesis

Journal: PLoS ONE

Article Title: HPRT-Deficiency Dysregulates cAMP-PKA Signaling and Phosphodiesterase 10A Expression: Mechanistic Insight and Potential Target for Lesch-Nyhan Disease?

doi: 10.1371/journal.pone.0063333

Figure Lengend Snippet: ( A & B ), immuno-blot and quantification analysis of p-Syn (Ser9), data show that the lower expression of phospho-PKA substrate and p-Syn (Ser9) in response to forskolin treatment in HPRT-deficient MN9D cells is restored in the presence of Papaverine (200 µM). Error bars represent mean ± SEM of duplicate measurements of two independent experiments (n = 4). The asterisks (*) represent statistical significance between forskolin treated cells without papaverine treatment (p<0.05, t-test). ( C & D ), immuno-blot and quantification analysis of PDE10A expression after transfection of HPRT-deficient MN9D cells with siRNA directed to the mouse PDE10 gene. The data show lower expression PDE10A protein in cells transfected with siRNA-PDE10A relative to cells transfected with scramble control siRNA (siRNA-CTL). Error bars represent mean ± SEM of duplicate measurements carried out independently twice (n = 4). The asterisks (*) represent the statistical significant (p<0.05, t-test) between the control (open bar, siRNA-CTL) and siRNA-PDE10A transfected cells (closed bar). This significant reduction of PDE10A protein contributes to the enhanced expression of phospho-PKA-substrates including p-Syn observed in ( E & F ). Error bars represent mean ± SEM of duplicate measurements of two independent experiments (n = 4). The asterisk (*) represents the statistical significant (p<0.05, t-test) between the siRNA-CTL and siRNA-PDE10A HPRT-deficient transfected MND9 cells treated with forskolin.

Article Snippet: 2×10 5 HPRT-deficient MN9D cells in 6 well-plate were exposed to 80 pmole of control (scramble siRNA) or PDE10A siRNA (directed to mouse PDE10A) from Santa cruz biotechnology and transfected according to the established transfection protocol ( http://datasheets.scbt.com/siRNA_protocol.pdf ).

Techniques: Expressing, Transfection, Control

Journal: PLoS ONE

Article Title: Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa

doi: 10.1371/journal.pone.0161035

Figure Lengend Snippet: Oligonucleotide primers used for PCR amplification.

Article Snippet: The membrane was next incubated with a monoclonal anti-PDE10A antibody (clone 1C9, recommended by the manufacturer for western blot; Origene Technologies, Rockville, MD) for 16 h at 4°C.

Techniques: Amplification

Journal: PLoS ONE

Article Title: Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa

doi: 10.1371/journal.pone.0161035

Figure Lengend Snippet: RNA isolated from bovine testis, testicular tetraploid (spermatocytes) and haploid (spermatids) germ cells was reverse transcribed and the transcripts encoding PDE10A variant X3 and X5 were amplified by PCR using specific primers. Amplification products obtained with spermatid RNA from 2 different bulls are shown. Similar results were obtained with RNA isolated from the testis, spermatocytes and spermatids from another bull. The expected size of the amplicons is 2367 bp and 2349 bp for variants X3 and X5, respectively. The position of molecular weight standards (bp) is shown on the left.

Article Snippet: The membrane was next incubated with a monoclonal anti-PDE10A antibody (clone 1C9, recommended by the manufacturer for western blot; Origene Technologies, Rockville, MD) for 16 h at 4°C.

Techniques: Isolation, Reverse Transcription, Variant Assay, Amplification, Molecular Weight

Journal: PLoS ONE

Article Title: Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa

doi: 10.1371/journal.pone.0161035

Figure Lengend Snippet: Bull testicular sections were processed for immunohistochemistry as detailed in Materials & Methods using a monoclonal anti-PDE10A antibody (3G9; Panel A, C, D, E, and F) or non-immune commercial mouse IgG (Panel B). Positive signal (arrows in all Panels) appears as a brownish precipitate. Panels A and B show 400X magnification and Panels C—F show 1000X magnification. Sc stands for primary spermatocytes, Sd stands for round spermatids, and Es stands for elongated spermatids.

Article Snippet: The membrane was next incubated with a monoclonal anti-PDE10A antibody (clone 1C9, recommended by the manufacturer for western blot; Origene Technologies, Rockville, MD) for 16 h at 4°C.

Techniques: Immunohistochemistry

Journal: PLoS ONE

Article Title: Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa

doi: 10.1371/journal.pone.0161035

Figure Lengend Snippet: Washed spermatozoa were subjected to nitrogen cavitation and the homogenate was centrifuged at low speed and then at high speed to separate raw membranes and cytosolic fractions as described in Materials & Methods. Equal amount of proteins from total cavitated sperm and from the membrane and cytosolic fractions were subjected to electrophoresis, transferred onto a PVDF membrane and probed with a PDE10A monoclonal antibody (clone 1C9). An anti-mouse IgG secondary antibody conjugated to horseradish peroxidase was then used and the positive signal was revealed with ChemiDoc MP Imaging system. Molecular weight standards (kDa) are indicated on the left.

Article Snippet: The membrane was next incubated with a monoclonal anti-PDE10A antibody (clone 1C9, recommended by the manufacturer for western blot; Origene Technologies, Rockville, MD) for 16 h at 4°C.

Techniques: Membrane, Electrophoresis, Imaging, Molecular Weight

Journal: PLoS ONE

Article Title: Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa

doi: 10.1371/journal.pone.0161035

Figure Lengend Snippet: Washed spermatozoa were fixed with formaldehyde and permeabilized with Triton X-100. They were next incubated overnight with a monoclonal anti-PDE10A antibody (clone 3G9; A-F) or a commercial non-immune mouse IgG at the same concentration (G-I). Panels A, D & G show the positive immunofluorescent signal obtained using a DyLight 594-conjugated secondary antibody; Panels E & H show the presence/absence of the acrosome (PNA-FITC labeling) on spermatozoa from the same field of D & G, respectively. Panels B, F & I are Phase contrast image of the same respective field (A, D & G). Panel C shows the superposition of Panels A & B. Magnification is 1000X. Note that PDE10A is detected in the acrosomal region of acrosome intact (AI) but not of acrosome reacted (AR) sperm (D-F). Some non-specific staining is observed in the midpiece of both AI and AR sperm incubated with non-immune mouse IgG (G-I). Identical PDE10A localization is obtained when the immunodetection experiment is performed in the absence (A) or presence (D) of PNA-FITC, or using a FITC-conjugated secondary antibody (not shown).

Article Snippet: The membrane was next incubated with a monoclonal anti-PDE10A antibody (clone 1C9, recommended by the manufacturer for western blot; Origene Technologies, Rockville, MD) for 16 h at 4°C.

Techniques: Incubation, Concentration Assay, Labeling, Staining, Immunodetection

Journal: PLoS ONE

Article Title: Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa

doi: 10.1371/journal.pone.0161035

Figure Lengend Snippet: Freshly ejaculated bull spermatozoa were washed, fixed with acrolein and pelleted in agarose. Sections of 50 μm were obtained and processed for immunodetection as described in Materials & Methods using monoclonal anti PDE10A antibody (A & B) or non-immune commercial mouse IgG (C & D). Sections were then post-fixed, embedded, and ultrathin sections were prepared and observed by transmission electron microscopy. Note that there is some non-specific signal in the midpiece region that is observed with both the PDE10A antibody and the non-immune IgG (arrowheads in A, B & C). Photographs were taken with magnification of 6800 X (A & C) and 9300 X (B & C). Acrosomal matrix (acr), plasma membrane (pm), and outer acrosomal membrane (oam; lining the acrosomal matrix) are indicated in the sperm head; mitochondria (mit), microtubules (mit) and outer dense fibers (odf) are indicated in the sperm midpiece. In all the panels, the bar scale represents 1 μm.

Article Snippet: The membrane was next incubated with a monoclonal anti-PDE10A antibody (clone 1C9, recommended by the manufacturer for western blot; Origene Technologies, Rockville, MD) for 16 h at 4°C.

Techniques: Immunodetection, Transmission Assay, Electron Microscopy, Clinical Proteomics, Membrane

Journal: PLoS ONE

Article Title: Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa

doi: 10.1371/journal.pone.0161035

Figure Lengend Snippet: A) Ejaculated bull spermatozoa were processed for Immunoprecipitation as described in Materials & Methods using a PDE10A monoclonal antibody and protein G-coupled sepharose. The immune-complex on the beads was next solubilized with sample buffer, subjected to electrophoresis and transferred onto PVDF membrane. Proteins were next probed with a monoclonal anti-PDE10A antibody and revealed with a secondary antibody conjugated to horseradish peroxidase, enhanced chemiluminescence, and film exposure. Molecular weight standards (kDa) are indicated on the left. B) Coverage of the PDE10A isoform X4 amino acid sequence by the trypsin generated peptides identified by LC-MS/MS. Sequence sections highlighted in yellow matched the peptides identified. Amino acids highlighted in green show variable post-translational modifications (Oxidation of M, Deamination of Q or N).

Article Snippet: The membrane was next incubated with a monoclonal anti-PDE10A antibody (clone 1C9, recommended by the manufacturer for western blot; Origene Technologies, Rockville, MD) for 16 h at 4°C.

Techniques: Immunoprecipitation, Electrophoresis, Membrane, Molecular Weight, Sequencing, Generated, Liquid Chromatography with Mass Spectroscopy

Journal: PLoS ONE

Article Title: Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa

doi: 10.1371/journal.pone.0161035

Figure Lengend Snippet: Intron-exon distribution deduced from the sequences obtained in our 5'-RLM (upper part) and 3'-RACE (lower part) experiments is presented. The numbers in the boxes refer to the number of nucleotides in the putative exons. The length of the introns is also indicated between the boxes. The position of the start of the ORFs in the variant X3 (isoform X2; MSHEPAEGGLDACDAS ), the variant X5 (isoform X4; MTFCGMARWY ), as well as the initial Methionine residue deduced from the different clones obtained in our 5’-RLM experiments that were similar to the variant X5 although with deletions in the 5’-UTR ( MQGVVYELNS ), are indicated. This ‘alternate’ start codon lies in the portion of the protein that is shared by all predicted PDE10A isoforms.

Article Snippet: The membrane was next incubated with a monoclonal anti-PDE10A antibody (clone 1C9, recommended by the manufacturer for western blot; Origene Technologies, Rockville, MD) for 16 h at 4°C.

Techniques: Variant Assay, Residue, Clone Assay