antibodies against ddx21  (Novus Biologicals)

Journal: The Journal of biological chemistry

Article Title: Pirh2-dependent DNA damage in neurons induced by the G-quadruplex ligand pyridostatin.

doi: 10.1016/j.jbc.2023.105157

Figure Lengend Snippet: Figure 8. Pirh2 downregulates DDX21 in cultured primary neurons. A, primary cortical neurons were transfected with the DDX21-NeonGreen construct. Cells were fixed and stained with antibodies against nucleolin and MAP2c and the DAPI dye. Note that DDX21-NeonGreen forms puncta. Scale bar rep- resents 5 μm. B, primary cortical neurons were transfected with DDX21-NeonGreen and treated with a vehicle or 2 μM pyridostatin (PDS) overnight. Cells were fixed, stained with antibodies against nucleolin and MAP2c, and imaged with a confocal microscope. The left panel is merged images (MAP2c, nucleolin, DDX21-NeonGreen, and DAPI). Scale bar represents 5 μm. The right three panels are zoomed in from the left panel to demonstrate that, in vehicle- treated neurons, nucleolin forms ring-like structures (the granular component of the nucleolus) and DDX21-NeonGreen forms puncta mostly within the nucleolin rings (the dense fibrillar component of the nucleolus). Note that PDS profoundly alters nucleolin- and DDX21-NeonGreen-positive foci. Scale bar represents 1 μm. C, colocalization of nucleolin and DDX21-NeonGreen was measured from (B). p = 0.014 (t test), 50 vehicle- and 50 PDS-treated neurons were analyzed from three experiments. D, primary cortical neurons were transfected with Neon-Green and BFP or with NeonGreen and BFP-Pirh2, fixed, and stained with antibodies against DDX21 and the SYTOX dye. Note that DDX21-positive puncta are absent in BFP-Pirh2-expressing cell (depicted with arrow). Scale bar represents 5 μm. E, fluorescence intensities of DDX21 were measured in BFP- and BFP-Pirh2-expressing neurons from (D). p = 0.001 (t test), 33 BFP-transfected and 44 BFP-Pirh2-transfected neurons were analyzed from three experiments. BFP, blue fluorescent protein; DAPI, 40,6-diamidino-2- phenylindole.

Article Snippet: Antibodies against DDX21 were from Proteintech (catalog no.: 10528-1-AP).

Techniques: Cell Culture, Transfection, Construct, Staining, Microscopy, Expressing

Journal: The Journal of biological chemistry

Article Title: Pirh2-dependent DNA damage in neurons induced by the G-quadruplex ligand pyridostatin.

doi: 10.1016/j.jbc.2023.105157

Figure Lengend Snippet: Figure 10. Model of Pirh2-dependent DNA damage in neurons induced by the G-quadruplex ligand pyridostatin (PDS). PDS alters transcription in cultured primary neurons, leading to changes in various neuron-specific and neuron-nonspecific pathways. The E3 ubiquitin ligase Pirh2 is upre- gulated and localizes to the nucleolus, resulting in DNA damage as evi- denced by γH2AX upregulation, downregulation of the helicase DDX21, and elevated G4-DNA levels.

Article Snippet: Antibodies against DDX21 were from Proteintech (catalog no.: 10528-1-AP).

Techniques: Cell Culture, Ubiquitin Proteomics

Journal: Viruses

Article Title: DEAD-Box RNA Helicase 21 (DDX21) Positively Regulates the Replication of Porcine Reproductive and Respiratory Syndrome Virus via Multiple Mechanisms

doi: 10.3390/v14030467

Figure Lengend Snippet: The effect of DDX21 on PRRSV replication. ( a , b ) iPAM cells were transfected with a Flag-tagged pDDX21 expression plasmid (3 μg) or empty vectors (3 μg) for 30 h and then infected with PRRSV (MOI = 0.1). At 12 hpi and 24 hpi, the cells were collected for use in qRT-PCR ( a ) and TCID 50 assay ( b ). ( c ) iPAM cells were transfected with one of three pDDX21-specific siRNAs (siDDX21-1, siDDX21-2, siDDX21-3) or negative control siRNA (NC) for 36 h. The interfering efficiency was measured by qRT-PCR (upper) and Western blotting (lower). ( d , e ) iPAM cells were transfected with siDDX21-3 or NC for 24 h and then infected with PRRSV (MOI = 0.1). At 12 hpi and 24 hpi, the cells were collected for use in qRT-PCR ( d ) and TCID 50 assay ( e ). All experiments were performed in triplicate, and the data are presented as the means ± SD of three independent experiments (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Rabbit polyclonal antibody against DDX21 (NB100-1718) was purchased from NOVUS (Shanghai, China).

Techniques: Transfection, Expressing, Plasmid Preparation, Infection, Quantitative RT-PCR, Negative Control, Western Blot

Journal: Viruses

Article Title: DEAD-Box RNA Helicase 21 (DDX21) Positively Regulates the Replication of Porcine Reproductive and Respiratory Syndrome Virus via Multiple Mechanisms

doi: 10.3390/v14030467

Figure Lengend Snippet: PRRSV infection promotes pDDX21 translocation from the nucleus to the cytoplasm. ( a , b ) iPAM cells were infected with PRRSV (MOI = 1). At 12, 24, and 36 hpi, these cells were collected for use in qRT-PCR to determine the number of mRNA copies of pDDX21 ( a ) or in Western blotting with antibodies against DDX21, PRRSV N protein, and β-actin ( b ). ( c , d ) iPAM cells were infected with increasing doses of PRRSV (0.01 MOI, 0.1 MOI, or 1.0 MOI). At 24 hpi, these cells were collected for use in qRT-PCR ( c ) and Western blotting ( d ) as described in ( a , b ), respectively. ( e ) iPAM cells were grown until they formed a monolayer on 100-mm plates and then infected with PRRSV (MOI = 0.1). These cells were collected at 24 hpi, and a nuclear cytosol fractionation assay was performed to detect the pDDX21 expression in the nucleus or cytoplasm. Data are presented as the means ± SD of three independent experiments (* p < 0.05; *** p < 0.001). The relative levels of pDDX21 in PRRSV-infected cells as compared with in mock-infected cells were analyzed by ImageJ software, and the ratio is displayed below the images as the fold change.

Article Snippet: Rabbit polyclonal antibody against DDX21 (NB100-1718) was purchased from NOVUS (Shanghai, China).

Techniques: Infection, Translocation Assay, Quantitative RT-PCR, Western Blot, Fractionation, Expressing, Software

Journal: Viruses

Article Title: DEAD-Box RNA Helicase 21 (DDX21) Positively Regulates the Replication of Porcine Reproductive and Respiratory Syndrome Virus via Multiple Mechanisms

doi: 10.3390/v14030467

Figure Lengend Snippet: PRRSV nsp1β upregulates DDX21 transcription and expression. ( a , b ) HEK-293T cells were transfected with 3 μg of plasmids expressing HA-tagged nsp1α, nsp1β, nsp4, nsp12, or N protein. At 30 h post-transfection, the cells were collected to detect the mRNA expression of DDX21 by qRT-PCR ( a ) or DDX21 protein expression by Western blotting ( b ). ( c – h ) HEK-293T cells ( c , d ) and MARC-145 cells ( e , f ) were transfected with increasing amounts (0.1875 μg, 0.375 μg, 0.75 μg, 1.5 μg, 3 μg) of plasmid encoding HA-tagged nsp1β or empty vector (3 μg). iPAM cells ( g , h ) were transfected with increasing amounts (0.375 μg, 0.75 μg, 1.5 μg, 3 μg) of plasmid encoding HA-tagged nsp1β or empty vector (3 μg). At 30 h post-transfection, the cells were collected for use in qRT-PCR ( c , e , g ) or Western blotting ( d , f , h ). All experiments were performed in triplicate. The data are shown as the means ± SD of three independent experiments (n.s, no significant differences; * p < 0.05; ** p < 0.01). The relative levels of DDX21 in experimentally transfected cells as compared with in empty vector-transfected cells were analyzed by ImageJ software, and the resulting ratio is displayed below the images as the fold change.

Article Snippet: Rabbit polyclonal antibody against DDX21 (NB100-1718) was purchased from NOVUS (Shanghai, China).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Software

Journal: Viruses

Article Title: DEAD-Box RNA Helicase 21 (DDX21) Positively Regulates the Replication of Porcine Reproductive and Respiratory Syndrome Virus via Multiple Mechanisms

doi: 10.3390/v14030467

Figure Lengend Snippet: nsp1β interacts with the C-terminal of pDDX21 ( a ) iPAM cells were infected with PRRSV (MOI = 0.5). At 36 hpi, these cells were lysed and immunoprecipitated with anti-nsp1β or anti-IgG monoclonal antibodies. WCL and IP complexes were analyzed by immunoblotting with anti-DDX21, anti-nsp1β, and anti-β-actin antibodies; ( b ) Schematic representation of WT pDDX21 and its truncation mutants. ( c ) HEK-293T cells were co-transfected with expression plasmids of Flag-tagged pDDX21-WT (3 μg) or its truncation mutants (3 μg) and HA-tagged nsp1β (3 μg). At 30 h post-transfection, these cells were lysed and immunoprecipitated with an anti-HA monoclonal antibody. WCL and IP complexes were analyzed by immunoblotting with anti-Flag, anti-HA, or anti-β-actin antibodies.

Article Snippet: Rabbit polyclonal antibody against DDX21 (NB100-1718) was purchased from NOVUS (Shanghai, China).

Techniques: Infection, Immunoprecipitation, Bioprocessing, Western Blot, Transfection, Expressing

Journal: Viruses

Article Title: DEAD-Box RNA Helicase 21 (DDX21) Positively Regulates the Replication of Porcine Reproductive and Respiratory Syndrome Virus via Multiple Mechanisms

doi: 10.3390/v14030467

Figure Lengend Snippet: pDDX21 downregulates IFN-β by impairing the activation IRF3 and p65 during PRRSV infection. ( a ) iPAM cells were transfected with siDDX21-3 or NC for 24 h and then infected with PRRSV (MOI = 0.5). At 12 hpi and 24 hpi, the cells were collected to detect pIFN-β mRNA via qRT-PCR. ( b , c ) iPAM cells were transfected with siDDX21-3 or NC for 24 h and then infected with PRRSV (MOI = 0.5). At 24 hpi, the cells were collected for use in a Western blotting analysis with the antibodies anti-DDX21, anti-phosphorylated IRF3, anti-total IRF3, anti-phosphorylated p65, anti-total p65, or anti-β-actin ( b ) or in qRT-PCR for detecting the mRNA expression levels of pDDX21, pISG15, pISG54, and pISG56 ( c ). All experiments were performed in triplicate. The data are presented as the means ± SD of three independent experiments (* p < 0.05; *** p < 0.001).

Article Snippet: Rabbit polyclonal antibody against DDX21 (NB100-1718) was purchased from NOVUS (Shanghai, China).

Techniques: Activation Assay, Infection, Transfection, Quantitative RT-PCR, Western Blot, Expressing