rabbit polyclonal anti ddx21  (Novus Biologicals)

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

Journal: Annals of neurology

Article Title: Dominant KPNA3 Mutations Cause Infantile-Onset Hereditary Spastic Paraplegia.

doi: 10.1002/ana.26228

Figure Lengend Snippet: FIGURE 2: Bar plot of karyopherin-α3-interacting proteins ranked by their respective connectivity quotient. Proteins are depicted on the y-axis by their respective symbols. The connectivity quotient for each protein is calculated as the number of links to proteins of the hereditary spastic paraplegia (HSP) module divided by the number of all links in the interactome. DDX21 displays a connectivity quotient of 0.33, indicating that 33% of all protein interactions of DDX21 are with proteins associated with the HSP module. Arrows indicate proteins chosen for functional analyses.

Article Snippet: Lysis of transfected HEK293T cells, immunopurification of EGFP-tagged recombinant proteins from lysates utilizing GFPTrapMagnetic Agarose beads (Chromotek), andWestern blotting analysis were essentially performed as described.24–26 After blotting, membranes were incubated with the following antibodies: anti-DDX21 rabbit polyclonal antibodies (Bethyl, #A300-627A, A300-628A, and A300-629A), anti-green fluorescent protein mouse monoclonal antibody (Covance, #MMS-118P-200), anti-NCBP1/CBP80 (D7Z2Z) rabbit monoclonal antibody (Cell Signalling, #24964), anti-NCBP2 rabbit polyclonal antibody (Bethyl, #A302-553A), anti-RCC1 740 Volume 90, No. 5 ANNALS of Neurology 15318249, 2021, 5, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1002/ana.26228 by E gyptian N ational Sti.

Techniques: Functional Assay

Journal: Annals of neurology

Article Title: Dominant KPNA3 Mutations Cause Infantile-Onset Hereditary Spastic Paraplegia.

doi: 10.1002/ana.26228

Figure Lengend Snippet: FIGURE 4: Impaired cargo binding of karyopherin-α3 variants. Recombinant karyopherin-α3 proteins were expressed in HEK293T cells and purified from cell lysates by immunoprecipitation techniques. Precipitated proteins were subjected to Western blot analysis using specific antibodies. (A) Bar graph indicating amounts of endogenous binding partners co-purifying with affinity- isolated WT and variant karyopherin-α3. Vertical lines indicate the SEM. Statistical analysis was performed via ANOVA (*p < 0.05; ***p < 0.001; n.s. = not significant; n = 7–20). (B) Representative examples of Western blots performed with GFP- Trap precipitates and antibodies recognizing RCC1, DDX21, NCBP1, and NCBP2. HEK293T cell lysates (10μg protein) were loaded for input lanes, and equal precipitate fractions were loaded into the remaining lanes. For better clarity, variants are referred to by their respective amino acid substitution only. (C) Representative Western blots performed with whole-cell lysates (input) and precipitates obtained from cells expressing EGFP-tagged wild-type karyopherin-α3 (WT) or EGFP alone and 2 distinct ataxin-3 antibodies. EGFP = enhanced green fluorescent protein; GFP = green fluorescent protein; WT = wild-type.

Article Snippet: Lysis of transfected HEK293T cells, immunopurification of EGFP-tagged recombinant proteins from lysates utilizing GFPTrapMagnetic Agarose beads (Chromotek), andWestern blotting analysis were essentially performed as described.24–26 After blotting, membranes were incubated with the following antibodies: anti-DDX21 rabbit polyclonal antibodies (Bethyl, #A300-627A, A300-628A, and A300-629A), anti-green fluorescent protein mouse monoclonal antibody (Covance, #MMS-118P-200), anti-NCBP1/CBP80 (D7Z2Z) rabbit monoclonal antibody (Cell Signalling, #24964), anti-NCBP2 rabbit polyclonal antibody (Bethyl, #A302-553A), anti-RCC1 740 Volume 90, No. 5 ANNALS of Neurology 15318249, 2021, 5, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1002/ana.26228 by E gyptian N ational Sti.

Techniques: Binding Assay, Recombinant, Immunoprecipitation, Western Blot, Isolation, Variant Assay, Expressing