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: 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

Journal: Cancer Medicine

Article Title: Genomic characterization of individuals presenting extreme phenotypes of high and low risk to develop tobacco‐induced lung cancer

doi: 10.1002/cam4.1500

Figure Lengend Snippet: Univariate and multivariate Cox regression analyses of PDE10A protein expression for recurrence‐free survival (RFS) and overall survival (OS) in patients with stage I–II NSCLC

Article Snippet: We used an anti‐human PDE10A antibody (Genetex) diluted 1:500.

Techniques: Expressing