rabbit polyclonal antibodies against flag pm020  (MBL International)

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: Screening PRRSV proteins for their potential interaction with pDDX21. ( a ) HEK-293T cells were co-transfected with pCAGGS-Flag-pDDX21 (3 μg) and HA-tagged PRRSV protein expression plasmids (3 μg). At 30 h post-transfection, the cells were lysed and immunoprecipitated with an anti-Flag monoclonal antibody. Whole-cell lysate (WCL) and IP complexes were analyzed by immunoblotting with antibodies against Flag, HA, or β-actin. ( b – f ) HEK-293T cells were co-transfected with pCAGGS-Flag-pDDX21 (3 μg) and HA-tagged nsp1α ( b ), nsp1β ( c ), nsp4 ( d ), nsp12 ( e ), or N protein ( f ) expression plasmids (3 μg). The cells were lysed at 30 h post-transfection and immunoprecipitated with an anti-HA monoclonal antibody. WCL and IP complexes were analyzed by immunoblotting with antibodies against Flag, HA, or β-actin. ( g ) iPAM cells were co-transfected with pCAGGS-Flag-pDDX21 (1 μg) and HA-tagged nsp1α, 1β, nsp4, nsp12, or N protein expression plasmids (1 μg). At 30 h post-transfection, the cells were fixed for an immunofluorescence assay analysis of pDDX21 and nsp1α, nsp1β, nsp4, nsp12, or N protein using primary antibodies (mouse anti-Flag and rabbit anti-HA), followed by secondary antibodies (AF594-conjugated donkey anti-rabbit and AF488-conjugated donkey anti-mouse). Nuclei were counterstained with DAPI. Fluorescent images were acquired with a confocal laser scanning microscope (Fluoviewver.3.1; Olympus, Japan).

Article Snippet: At 30 h post-transfection, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature (RT), permeabilized with methanol for 15 min, blocked in 10% bovine serum albumin for 30 min, and then incubated with mouse monoclonal antibody against HA (M180-3) (dilution 1:400, MBL, Beijing, China) and rabbit polyclonal antibodies against Flag (PM020) (dilution 1:400, MBL, Beijing).

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, Immunofluorescence, Laser-Scanning Microscopy

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: At 30 h post-transfection, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature (RT), permeabilized with methanol for 15 min, blocked in 10% bovine serum albumin for 30 min, and then incubated with mouse monoclonal antibody against HA (M180-3) (dilution 1:400, MBL, Beijing, China) and rabbit polyclonal antibodies against Flag (PM020) (dilution 1:400, MBL, Beijing).

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 stabilizes the expression of PRRSV nsp1α, nsp1β, and N protein. ( a – e ) iPAM cells were co-transfected with increasing concentrations (0.5 µg, 1.0 µg, or 2.0 µg) of Flag-tagged pDDX21 expression plasmid and 1.0 µg of plasmid encoding HA-tagged nsp1α ( a ), nsp1β ( b ), nsp4 ( c ), nsp12 ( d ), or N protein ( e ). At 30 h post-transfection, the cells were collected for use in a Western blotting analysis with antibodies against Flag, HA, or β-actin. The relative levels of nsp1α, nsp1β, nsp4, nsp12, and N protein in transfected cells as compared with the levels in empty vector-transfected cells were analyzed by ImageJ software, and the resulting ratio is displayed below the images as fold change.

Article Snippet: At 30 h post-transfection, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature (RT), permeabilized with methanol for 15 min, blocked in 10% bovine serum albumin for 30 min, and then incubated with mouse monoclonal antibody against HA (M180-3) (dilution 1:400, MBL, Beijing, China) and rabbit polyclonal antibodies against Flag (PM020) (dilution 1:400, MBL, Beijing).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Software