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: 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