primary antibodies to detect pdcov nucleoprotein (n)  (Alpha Diagnostics)

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

Journal: bioRxiv

Article Title: The porcine deltacoronavirus replication organelle comprises double membrane vesicles and zippered endoplasmic reticulum with double membrane spherules

doi: 10.1101/719443

Figure Lengend Snippet: Dynamics of PDCoV OH-FD22 replication in LLC-PK1 cells. ( A ) LLC-PK1 cells were infected with PDCoV and at the indicated time points, RNA was harvested and reverse transcribed to cDNA. Copy number of cDNA was quantified by 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 time points 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 time points, cell culture media was harvested. The titer of progeny virus was determined by TCID 50 . Mean and standard deviation from three independent replicates are shown.

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

Techniques: Infection, Standard Deviation, Western Blot, Molecular Weight, Cell Culture

Journal: bioRxiv

Article Title: The porcine deltacoronavirus replication organelle comprises double membrane vesicles and zippered endoplasmic reticulum with double membrane spherules

doi: 10.1101/719443

Figure Lengend Snippet: PDCoV RNA synthesis can be 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 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, membranes were incubated with primary antibodies to detect PDCoV nucleoprotein (N) (Alpha Diagnostic International) and actin (Abcam) diluted in blocking buffer.

Techniques: Infection, Incubation, Staining

Journal: bioRxiv

Article Title: The porcine deltacoronavirus replication organelle comprises double membrane vesicles and zippered endoplasmic reticulum with double membrane spherules

doi: 10.1101/719443

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

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

Techniques: Infection, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: The porcine deltacoronavirus replication organelle comprises double membrane vesicles and zippered endoplasmic reticulum with double membrane spherules

doi: 10.1101/719443

Figure Lengend Snippet: DMVs and zippered ER with double membrane spherules are present from 6 to 24 hours post infection. LLC-PK1 cells were infected with PDCoV and at 6 (A and B) or 24 (C and D) hours post infection, cells were fixed with glutaraldehyde and processed for electron microscopy. Virions in vesicles are indicated with white arrows, DMVs are indicated with black 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, membranes were incubated with primary antibodies to detect PDCoV nucleoprotein (N) (Alpha Diagnostic International) and actin (Abcam) diluted in blocking buffer.

Techniques: Infection, Electron Microscopy