rabbit anti ddx21  (Novus Biologicals)

Journal: Cell reports

Article Title: TRMT1L-catalyzed m 2 2 G27 on tyrosine tRNA is required for efficient mRNA translation and cell survival under oxidative stress

doi: 10.1016/j.celrep.2024.115167

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Article Snippet: Rabbit anti-DDX21 Antibody , Bethyl , Cat#A300-627A; RRID: AB_513601.

Techniques: Recombinant, Membrane, Reverse Transcription, Protease Inhibitor, Magnetic Beads, Staining, Mutagenesis, Silver Staining, BIA-KA, RNA Sequencing, Plasmid Preparation, Software

Journal: Nature

Article Title: DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis

doi: 10.1038/s41586-020-2041-2

Figure Lengend Snippet: a, b, Immunofluorescence staining of endogenous KU86 (a) and DNA-PKcs (b) in U2OS cells. DDX21 RNA helicase is used as a positive control for nucleoli. The CSK buffer contains Triton X-100 for pre-extraction before fixation (see Methods). When indicated, the cells were treated with 50 nM ActD for 1 h before pre-extraction, fixation and staining. c, Localization of ectopically expressed GFP-tagged KU70 in mouse ES cells. a–c, n = 3 biologically independent experiments. d, U3 ChIRP-qRT–PCR analysis from HeLa cells. Enrichment levels, relative to input samples, of the U3, 7SK, 18S, and RMRP RNAs were assessed from experimental (−RNase A) or control (+RNase A) ChIRP samples. Data are from two independent biological replicates. e, DNA-PK was also recovered from U3 ChIRP-MS in IMR90 cells. Peptide spectral match (PSM) counts for control (RNase A) and experimental (U3) samples are shown. n = 2 biological replicates.

Article Snippet: Fixed cells were then incubated with primary antibodies in 3% BSA for 1 h at 25 °C, including mouse anti-human KU86 (ThermoFisher, MA5–12933, 1:100), rabbit anti-human DDX21 (Novus, NB100–1718, 1:500) or anti-DNA-PKcs (ThermoFisher, Ab-4(cocktail)), followed by fluorophore-conjugated secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit, Alexa Fluor 594-conjugated anti-rabbit, and cyanine3-conjugated anti-mouse, Invitrogen, 1:500) for 1 h at room temperature.

Techniques: Immunofluorescence, Staining, Positive Control, Extraction, Quantitative RT-PCR, Control

Journal: Frontiers in Microbiology

Article Title: DEAD-box RNA helicase 21 interacts with porcine circovirus type 2 Cap protein and facilitates viral replication

doi: 10.3389/fmicb.2024.1298106

Figure Lengend Snippet: DDX21 relocates from the nucleolus to the cytoplasm induced by PCV2 infection. (A) Immunofluorescence analysis of the subcellular localization of DDX21 during PCV2 infection. PK-15 cells were infected with PCV2 at a MOI of 1.0 for 24, 48, 72 h post-infection (hpi). Cells were fixed and stained with mouse mAb against Cap, rabbit anti-DDX21 antibody, FITC-labeled goat anti-mouse IgG, and Alexa Fluor 546-conjugated donkey anti-rabbit IgG. PK-15 cells were observed under a confocal microscope . Nuclei were stained with DAPI. Scale bar, 10 μm. (B) The proportion of co-localization of PCV2 Cap and DDX21 proteins was analyzed using ImageJ software at 24, 48, and 72 hpi. (C) The nuclear and cytoplasmic fractions were extracted after PK-15 cells infected with PCV2 at a MOI of 1.0. At 48 and 72 hpi, the protein samples were prepared and analyzed by western blotting using antibodies against PCV2 Cap and DDX21. Histone H3 and β-tubulin served as fractionation quality controls. (D) The DDX21 protein band intensity was analyzed using ImageJ software at 48, 72 hpi. Data are presented as means ± SD of three independent experiments. * p < 0.05.

Article Snippet: Rabbit mAb against DDX21 (ab182156) was purchased from Abcam (Cambridge, MA).

Techniques: Infection, Immunofluorescence, Staining, Labeling, Microscopy, Software, Western Blot, Fractionation

Journal: Frontiers in Microbiology

Article Title: DEAD-box RNA helicase 21 interacts with porcine circovirus type 2 Cap protein and facilitates viral replication

doi: 10.3389/fmicb.2024.1298106

Figure Lengend Snippet: Cap interacts directly with DDX21. (A) Immunoprecipitation analysis. PK-15 cells were infected with PCV2 at a MOI of 1.0 for 48 h and cell lysates were immunoprecipitated with purified anti-Cap IgG mAb, followed by immunoblotting with anti-Cap, anti-DDX21, and anti-β-actin antibodies. Besides, the PCV2-infected PK-15 cell lysates were further immunoprecipitated with purified anti-Rep IgG mAb, followed by immunoblotting with anti-Rep, anti-DDX21, and anti-β-actin antibodies . (B) PK-15 cells were transfected with an empty vector and FLAG-Cap for 48 h. (C,D) Co-immunoprecipitation analysis. HEK293T cells were co-transfected with Myc-Cap and FLAG-DDX21 for 48 h, and then the cell lysates were immunoprecipitated with Flag beads (B,C) or anti-Myc-purified IgG (D) . (E) GST pull-down assays. GST or GST-DDX21 proteins were immobilized on GST beads and incubated with His-sumo-Cap. GST or GST-DDX21 proteins in the GST pull-down assays were examined using immunoblotting with anti-His, and anti-GST antibodies, respectively. GST, glutathione-S-transferase.

Article Snippet: Rabbit mAb against DDX21 (ab182156) was purchased from Abcam (Cambridge, MA).

Techniques: Immunoprecipitation, Infection, Purification, Western Blot, Transfection, Plasmid Preparation, Incubation

Journal: Frontiers in Microbiology

Article Title: DEAD-box RNA helicase 21 interacts with porcine circovirus type 2 Cap protein and facilitates viral replication

doi: 10.3389/fmicb.2024.1298106

Figure Lengend Snippet: Binding domain identification of Cap with DDX21. HEK293T cells were co-transfected with plasmids containing full-length PCV2 Cap or truncated mutants fused with a GFP-, or FLAG-GST tag, along with FLAG-DDX21 plasmid for 48 h; the cell lysate extracts were immunoprecipitated with Flag beads (A) or anti-GFP purified IgG (B) , or pulled-down with glutathione S-transferase (GST) beads (C) and then detected by western blotting using the indicated antibodies. (D,E) The nuclear localization signals (NLSs) within capsid protein of porcine circovirus type 1, 2, 3, 4 and circoviruses from other species were responsible for binding to DDX21. HEK293T cells were cotransfected with plasmids encoding NLSs of PCV1, 2, 3, 4 (D) and circoviruses from terrestrial, aquatic and avian species, including pigs, canaries, canines, minks, dragonflies, pigeons, ducks, bats, geese, and parrots (E) , along with FLAG-DDX21; cell lysates were subjected to immunoprecipitation and immunoblotting using the indicated antibodies.

Article Snippet: Rabbit mAb against DDX21 (ab182156) was purchased from Abcam (Cambridge, MA).

Techniques: Binding Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Purification, Western Blot

Journal: Frontiers in Microbiology

Article Title: DEAD-box RNA helicase 21 interacts with porcine circovirus type 2 Cap protein and facilitates viral replication

doi: 10.3389/fmicb.2024.1298106

Figure Lengend Snippet: 763 GSRSNRFQNK 772 of DDX21 is crucial for binding to Cap. (A) Schematic representation of the NTD, Helicase D, and CTD of DDX21 and their truncation mutants used in this study. (B–D) The DDX21-CTD-(582-784aa) interacted with Cap. HEK293T cells were co-transfected with expression plasmids GFP-DDX21-WT or its serial GFP-DDX21 truncated mutants M1 to M6, together with FLAG-Cap or FLAG-GST-Cap plasmid. The cell lysate extracts were immunoprecipitated or GST pulled-down followed by western blotting using the indicated antibodies. (E,F) Identification the critical amino acids of DDX21-CTD essential for interaction with Cap. HEK293T cells were co-transfected with DDX21-WT or DDX21 truncated mutants M3, M7 to M9, along with FLAG-Cap plasmid, and the cell lysate extracts were immunoprecipitated followed by western blotting using the indicated antibodies.

Article Snippet: Rabbit mAb against DDX21 (ab182156) was purchased from Abcam (Cambridge, MA).

Techniques: Binding Assay, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Frontiers in Microbiology

Article Title: DEAD-box RNA helicase 21 interacts with porcine circovirus type 2 Cap protein and facilitates viral replication

doi: 10.3389/fmicb.2024.1298106

Figure Lengend Snippet: DDX21 promotes the replication of PCV2. (A,C) Immunoblotting of the proteins Cap, Rep, DDX21, FLAG, and β-actin in DDX21 -silenced or DDX21-overexpressing PK-15 cells. The cells were infected with PCV2 at a MOI of 1.0, and shCON-transfected or empty vector-transfected cells served as negative controls. (B,D) TCID 50 values of PCV2 in samples from (A,C) . Viral stocks were harvested at 48, 60, 72 hpi. Data are presented as mean ± SD from three independent biological experiments. ns, not significant; * p < 0.05.

Article Snippet: Rabbit mAb against DDX21 (ab182156) was purchased from Abcam (Cambridge, MA).

Techniques: Western Blot, Infection, Transfection, Plasmid Preparation

Journal: Frontiers in Microbiology

Article Title: DEAD-box RNA helicase 21 interacts with porcine circovirus type 2 Cap protein and facilitates viral replication

doi: 10.3389/fmicb.2024.1298106

Figure Lengend Snippet: List of primers adopted in the study.

Article Snippet: Rabbit mAb against DDX21 (ab182156) was purchased from Abcam (Cambridge, MA).

Techniques:

Journal: The Journal of Immunology Author Choice

Article Title: The RNA-Splicing Ligase RTCB Promotes Influenza A Virus Replication by Suppressing Innate Immunity via Interaction with RNA Helicase DDX1

doi: 10.4049/jimmunol.2200799

Figure Lengend Snippet: Protein–protein interaction between DDX1 and DDX21 was impeded by RTCB. (A) HeLa cells were cotransfected with Myc-RTCB and HA-DDX1. The localization of RTCB (green) and DDX1 (red) was assessed using confocal microscopy. DAPI was used to stain the nuclei (blue). (B) A549 cells from three 10-cm dishes were lysed with 500 μl IP lysis buffer, and immunoprecipitation was performed using anti-RTCB, anti-DDX1, or control IgG Abs. The pull-down products were analyzed using Western blotting. (C) HEK293T cells were cotransfected with Myc-RTCB and HA-DDX1. The co-IP experiment was performed using an anti-Myc or an anti-HA Ab, followed by Western blotting. (D and E) RTCBWT and RTCBKO A549 cells were cultured in 10-cm dishes and lysed with 500 μl IP lysis buffer, followed by immunoprecipitation assay with anti-DDX1, anti-DDX21, or control IgG; the pull-down products were analyzed using Western blotting. (F and G) HEK293T cells were cotransfected with HA-DDX1, Flag-DDX21, Myc-DHX36, or an empty vector, as well as with increasing quantities of Myc-RTCB for 24 h. The co-IP experiment was performed using an anti-HA or an anti-Flag Ab, followed by Western blotting. In each Western blot assay, the blots originated either from the same membrane or from reloading the same quantity of lysates in the same experiments. (H) The proposed model of the action of RTCB on the conformation of the DDX1-DDX21-DDX36 complex.

Article Snippet: Abs The Abs used in this study were anti-RTCB rabbit polyclonal Ab (Cat no. 19809-1-AP; Proteintech), anti-DDX1 rabbit polyclonal Ab (Cat no. 11357-1-AP; Proteintech), anti-DDX21 rabbit polyclonal Ab (Cat no. 10528-1-AP; Proteintech), anti-DHX36 rabbit polyclonal Ab (Cat no. 13159-1-AP; Proteintech), anti-Flag mouse mAb (Cat no. F1804; Sigma), anti-HA rabbit polyclonal Ab (Cat no. 51064-2-AP; Proteintech), anti-GAPDH mouse mAb (Cat no. 60004-1-Ig; Proteintech), as well as Alexa Fluor 488–conjugated AffiniPure goat anti-mouse (Cat no. GM200G-02C; Sungene Biotech, Tianjin, China) and Alexa Fluor 594–conjugated AffiniPure goat anti-rabbit (Cat no. GR200G-43C; Sungene Biotech) secondary Abs.

Techniques: Confocal Microscopy, Staining, Lysis, Immunoprecipitation, Control, Western Blot, Co-Immunoprecipitation Assay, Cell Culture, Plasmid Preparation, Membrane