Journal: Science Translational Medicine

Article Title: A degradation fragment of type X collagen is a real-time marker for bone growth velocity

doi: 10.1126/scitranslmed.aan4669

Figure Lengend Snippet: Identification and subunit characterization of CXM marker . ( A ) Western blots of umbilical cord serum, adult serum, and recombinant full-length human type X collagen (rCOLX) (positive control). Equivalent blots of 4 to 12% gels were probed with antibodies to the noncollagenous NC2 domain (left panel), collagen helix (center panel), and noncollagenous NC1 domain (right panel). Fourth panel: Representative Coomassie stain of serum proteins present in cord and adult lanes. ( B ) Left panel: Western blot of immunoprecipitated collagen X marker (CXM) eluted at pH 7.0 versus pH 2.5, separated on a 12% gel, and probed with a pAb (USCNK) to the NC1 domain. Right panel: Recombinant trimeric NC1 (rNC1) separated by SDS–polyacrylamide gel before (left lane) or after (right lane) pH 2.5 treatment and stained for protein. std refers to molecular mass standards.

Article Snippet: Aves Labs Inc. prepared and purified a chicken pAb to the mouse rNC1 sequence above.

Techniques: Marker, Western Blot, Recombinant, Positive Control, Staining, Immunoprecipitation

Journal: Science Translational Medicine

Article Title: A degradation fragment of type X collagen is a real-time marker for bone growth velocity

doi: 10.1126/scitranslmed.aan4669

Figure Lengend Snippet: Marker decreases with age and is detected in human urine and mouse blood . Western blots of CXM aptoprecipitated with SOMA1and probed with X34 mAb from ( A ) serum of individuals of increasing ages (0 year, umbilical cord serum) or (B ) matched urine and serum samples from a 2-month-old infant (Vol, volume of sample; Exp, exposure time for autoradiography). ( C ) Aptoprecipitated trimeric markers from human serum (CXM) or mouse serum (Cxm) probed with pAbs raised against their respective recombinant NC1 domains and compared to Coomassiestained gels of the same recombinant proteins (rNC1).

Article Snippet: Aves Labs Inc. prepared and purified a chicken pAb to the mouse rNC1 sequence above.

Techniques: Marker, Western Blot, Autoradiography, Recombinant

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: The Niemann-Pick C1 gene interacts with a high-fat diet to promote weight gain through differential regulation of central energy metabolism pathways

doi: 10.1152/ajpendo.00369.2016

Figure Lengend Snippet: Amounts of liver SREBP-1 and SREBP-2 protein. A: representative Western blot analysis of liver SREBP-1 protein and β-actin for Npc1+/+ and Npc1+/− mice fed a LFD or HFD at 30 wk. B: amounts of liver SREBP-1 protein adjusted for β-actin and normalized to amounts of liver SREBP-1 protein for Npc1+/+ mice fed a LFD. C: representative Western blot analysis of liver SREBP-2 protein and β-actin for Npc1+/+ and Npc1+/− mice fed a LFD or HFD at 20 wk. D: amounts of liver SREBP-2 protein adjusted for β-actin and normalized to the amounts of liver SREBP-2 protein for Npc1+/+ mice fed a LFD. Values are expressed as means ± SE of six mice per group. *P < 0.05 compared with Npc1+/+ mice fed a LFD.

Article Snippet: The primary antibodies to detect mature sterol regulatory element-binding protein-1 (SREBP-1) and sterol regulatory element-binding protein-2 (SREBP-2) protein were purchased from Cayman Chemical (10007663) and Novus Biologicals (100–2215), respectively.

Techniques: Western Blot

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: The Niemann-Pick C1 gene interacts with a high-fat diet to promote weight gain through differential regulation of central energy metabolism pathways

doi: 10.1152/ajpendo.00369.2016

Figure Lengend Snippet: RNA microarray of livers from Npc1 +/+ and Npc1 +/− mice fed a HFD

Article Snippet: The primary antibodies to detect mature sterol regulatory element-binding protein-1 (SREBP-1) and sterol regulatory element-binding protein-2 (SREBP-2) protein were purchased from Cayman Chemical (10007663) and Novus Biologicals (100–2215), respectively.

Techniques: Microarray

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: The Niemann-Pick C1 gene interacts with a high-fat diet to promote weight gain through differential regulation of central energy metabolism pathways

doi: 10.1152/ajpendo.00369.2016

Figure Lengend Snippet: Schematic representation of the proposed Npc1 gene-diet metabolic pathway that promotes weight gain. A: dietary saturated fatty acids have been reported to increase expression of the Pgc1a and Pgc1b genes and the amounts of encoded PGC-1α and PGC1-β proteins. B: the PGC-1α and PGC1-β proteins serve as transcriptional coactivators for the nuclear receptor LXR (activated in the presence of oxysterol) that forms a heterodimer with RXR (activated in the presence of retinoic acid) to increase expression of the Srebp1 gene and the amounts of encoded precursor SREBP-1 protein. C: decreased Npc1 gene dosage and decreased amounts of encoded NPC1 protein has been reported to impair feedback inhibition of the SREBP pathway characterized by an increased amount of mature SREBP-1 protein that serves as a transcription factor. D: the increased amounts of PGC-1β protein (but not PGC-1α protein) have also been reported to serve as a transcriptional coactivator for the increased amounts of mature SREBP-1 protein to increase expression of target genes encoding proteins that regulate increased glycolysis and lipogenesis but also decreased expression of the Ppara gene and the amounts of encoded PPARα protein. E: the PGC-1α protein (but not PGC-1β protein) serves as a transcriptional coactivator for the decreased amounts of PPARα protein that forms a heterodimer with RXR and decreases expression of target genes. F: these PPARα target genes encode proteins that regulate triacylglycerol lipolysis and fatty acid β-oxidation. Therefore, the proposed Npc1 gene-diet interaction metabolic pathway promotes weight gain through increased glycolysis and lipogenesis in addition to decreased triacylglycerol lipolysis and fatty acid β-oxidation. PGC-1α, peroxisome proliferator-activated receptor gamma coactivator-1α; PGC-1β peroxisome-proliferator activated receptor gamma coactivator-1β; LXR, liver X receptor; RXR, retinoid X receptor; LXRE, liver X receptor response element; SREBP-1, sterol regulatory element-binding protein-1; SREBP, sterol regulatory element-binding protein response element; PPARα, peroxisome-proliferator activated receptor alpha; PPRE, peroxisome-proliferator activated receptor response element.

Article Snippet: The primary antibodies to detect mature sterol regulatory element-binding protein-1 (SREBP-1) and sterol regulatory element-binding protein-2 (SREBP-2) protein were purchased from Cayman Chemical (10007663) and Novus Biologicals (100–2215), respectively.

Techniques: Expressing, Inhibition, Binding Assay

Journal: Viruses

Article Title: The Porcine Deltacoronavirus Replication Organelle Comprises Double-Membrane Vesicles and Zippered Endoplasmic Reticulum with Double-Membrane Spherules

doi: 10.3390/v11111030

Figure Lengend Snippet: Dynamics of porcine deltacoronavirus (PDCoV) OH-FD22 replication in LLC-PK1 cells. ( A ) LLC-PK1 cells were infected with PDCoV and at the indicated timepoints, RNA was harvested and reverse transcribed to cDNA. The copy number of cDNA was quantified by quantitative polymerase chain reaction (qPCR) using a standard curve and normalized to mock-infected cells. Mean and standard deviation of three independent replicates are shown. ( B ) LLC-PK1 cells were PDCoV-infected or mock-infected. Total cell lysate was harvested at the stated timepoints and viral nucleoprotein detected (Anti-N) by western blot. Actin (Anti-actin) was used as a loading control. Molecular weight markers are shown. Blot representative of three independent repeats. ( C ) LLC-PK1 cells were infected with PDCoV and at the indicated timepoints and cell culture media was harvested. The titer of progeny virus was determined by tissue culture infectious dose 50 (TCID 50 ). Mean and standard deviation from three independent replicates are shown.

Article Snippet: After blocking in 5% milk in PBS-Tween 20 (PBS-T), membranes were incubated with primary antibodies to detect PDCoV nucleoprotein (N) (Alpha Diagnostic International) and actin (Abcam, Cambridge, UK) diluted in blocking buffer.

Techniques: Infection, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation, Western Blot, Control, Molecular Weight, Cell Culture, Virus

Journal: Viruses

Article Title: The Porcine Deltacoronavirus Replication Organelle Comprises Double-Membrane Vesicles and Zippered Endoplasmic Reticulum with Double-Membrane Spherules

doi: 10.3390/v11111030

Figure Lengend Snippet: PDCoV-associated dsRNA can be detected from 4 h post-infection (hpi). LLC-PK1 cells were infected or mock-infected. At the stated timepoints, cells were fixed and labelled with anti-dsRNA (green) and anti-N (red). Nuclei were stained with DAPI (blue) and scale bar indicates 10 μm. Images are representative of three independent replicates.

Article Snippet: After blocking in 5% milk in PBS-Tween 20 (PBS-T), membranes were incubated with primary antibodies to detect PDCoV nucleoprotein (N) (Alpha Diagnostic International) and actin (Abcam, Cambridge, UK) diluted in blocking buffer.

Techniques: Infection, Staining

Journal: Viruses

Article Title: The Porcine Deltacoronavirus Replication Organelle Comprises Double-Membrane Vesicles and Zippered Endoplasmic Reticulum with Double-Membrane Spherules

doi: 10.3390/v11111030

Figure Lengend Snippet: PDCoV RNA synthesis was detected from 3 hpi. LLC-PK1 cells were infected or mock-infected. Thirty minutes prior to the indicated fixation time, cells were incubated with 2 mM 5-Bromouridine (BrU) and 15 µM actinomycin D (ActD). Mock cells were incubated with (+ActD) and without (−ActD) actinomycin D as indicated. Cells were fixed and RNA containing BrU were detected using an anti-BrdU antibody (green). Nuclei were stained with DAPI (blue), scale bar indicates 10 μm. Images representative of three independent replicates.

Article Snippet: After blocking in 5% milk in PBS-Tween 20 (PBS-T), membranes were incubated with primary antibodies to detect PDCoV nucleoprotein (N) (Alpha Diagnostic International) and actin (Abcam, Cambridge, UK) diluted in blocking buffer.

Techniques: Infection, Incubation, Staining

Journal: Viruses

Article Title: The Porcine Deltacoronavirus Replication Organelle Comprises Double-Membrane Vesicles and Zippered Endoplasmic Reticulum with Double-Membrane Spherules

doi: 10.3390/v11111030

Figure Lengend Snippet: Sites of PDCoV RNA synthesis were associated with viral N protein, but not the endoplasmic reticulum (ER). LLC-PK1 cells were infected or mock-infected. Thirty minutes prior to fixation, cells were incubated with 2 mM 5-Bromouridine (BrU) and 15 μM actinomycin D. Cells were fixed and labelled with anti-BrdU (green) and either anti-N or anti-ER antibodies (red). Nuclei were stained with DAPI (blue), scale bar indicates 10 µm.

Article Snippet: After blocking in 5% milk in PBS-Tween 20 (PBS-T), membranes were incubated with primary antibodies to detect PDCoV nucleoprotein (N) (Alpha Diagnostic International) and actin (Abcam, Cambridge, UK) diluted in blocking buffer.

Techniques: Infection, Incubation, Staining

Journal: Viruses

Article Title: The Porcine Deltacoronavirus Replication Organelle Comprises Double-Membrane Vesicles and Zippered Endoplasmic Reticulum with Double-Membrane Spherules

doi: 10.3390/v11111030

Figure Lengend Snippet: PDCoV RO is made up of double-membrane vesicles (DMVs) and zippered ER with double-membrane spherules. LLC-PK1 cells were mock-infected ( A ) or infected with PDCoV ( B – F ). At 8 hpi, cells were fixed with glutaraldehyde and processed for transmission electron microscopy. Virions in vesicles are indicated with black arrows, DMVs are indicated with white arrows, and regions of zippered ER with spherules are indicated with black brackets. Scale bars indicate 1 µm ( A , B ) or 500 nm ( C – F ).

Article Snippet: After blocking in 5% milk in PBS-Tween 20 (PBS-T), membranes were incubated with primary antibodies to detect PDCoV nucleoprotein (N) (Alpha Diagnostic International) and actin (Abcam, Cambridge, UK) diluted in blocking buffer.

Techniques: Membrane, Infection, Transmission Assay, Electron Microscopy

Journal: Viruses

Article Title: The Porcine Deltacoronavirus Replication Organelle Comprises Double-Membrane Vesicles and Zippered Endoplasmic Reticulum with Double-Membrane Spherules

doi: 10.3390/v11111030

Figure Lengend Snippet: DMVs and zippered ER with double-membrane spherules were present from 6 hpi to 24 hpi. LLC-PK1 cells were infected with PDCoV, and at 6 hpi ( A , B ) or 24 hpi ( C , D ), cells were fixed with glutaraldehyde and processed for electron microscopy. Virions in vesicles are indicated with black arrows, DMVs are indicated with white arrows, and regions of zippered ER with spherules are indicated with black brackets. Scale bars indicate 200 nm ( A ) or 500 nm ( B – D ).

Article Snippet: After blocking in 5% milk in PBS-Tween 20 (PBS-T), membranes were incubated with primary antibodies to detect PDCoV nucleoprotein (N) (Alpha Diagnostic International) and actin (Abcam, Cambridge, UK) diluted in blocking buffer.

Techniques: Membrane, Infection, Electron Microscopy