ddx21 antibody - bsa free  (Bio-Techne corporation)

Journal: Nature Communications

Article Title: Uncoupling of mTORC1 from E2F activity maintains DNA damage and senescence

doi: 10.1038/s41467-024-52820-6

Figure Lengend Snippet: a Comparison of nucleolar area between cycling cells and 5-day p16 arrested cells (>1000 cells for each condition). b Evaluation of transcriptional activity in G1-phase cycling cells or in 3-day arrested cells. Cells were pulsed with 5-Ethynyl Uridine (EU; 100 µM) for one hour prior to fixation and EU nuclear intensity measured by fluorescence imaging (>1000 cells for each condition). c Evaluation of transcriptional activity in cells arrested for 3 days in either full growth media, starvation media lacking serum and growth factors, or full growth media + Torin2 (100 nM). Cells were pulsed with 5-Ethynyl Uridine (EU; 100 µM) for one hour prior to fixation and EU nuclear intensity measured by fluorescence imaging (>1000 cells for each condition). d Representative immunofluorescence images of 3-day p16 arrested cells stained for 53BP1 to visualize DNA damage and nucleolin to visualize nucleoli. 53BP1 puncta are frequently observed in the perinucleolar area (arrows). e Manual quantification of the number of perinucleolar DNA damage puncta per cells in G1-phase cycling cells and 3-day arrested cells ( n = 250 cells per condition). f Three-day p16 arrested cells were fixed following a one-hour EU (100 µM) pulse and stained for γ-H2AX by immunofluorescence. Cells were binned into 5 groups by EU nuclear intensity and the average γ-H2AX intensity was calculated for each group of cells (bin 1, n = 1155; bin 2, n = 5252; bin 3, n = 3141; bin 4, n = 855; bin 5, n = 201). g Representative images of cycling cells and 3-day arrested cells stained for Ddx21 by immunofluorescence. h quantification of the ratio of Ddx21 nucleolar fluorescence intensity to Ddx21 nucleoplasmic fluorescence intensity in G1 cycling cells and 3-day arrested cells treated with DMSO or with CX-5461 (5 µM) (>1000 cells for each condition). Kolmogorov–Smirnov test was used for analysis in ( a – c and h ). *** indicate p < 2.2 × 10 −16 .

Article Snippet: Primary antibodies used in this study were rabbit anti-phospho-H2AX (1:500, Cell Signaling Technologies #9718), rabbit anti-53BP1 (1:500, Cell Signaling Technologies #4937), rabbit anti-phospho-Rb (Ser807/811) (1:2500, Cell Signaling Technology #8516), rabbit anti-phospho-S6 Ribosomal Protein (1:500, Cell Signaling Technology #2215), rabbit anti-cGAS (1:500, Cell Signaling Technology #15102), rabbit phospho-Chk2 (Thr68) (1:500, Cell Signaling Technology # #2197), rabbit anti-cyclin D1 (1:500, Thermo-Fisher Scientific #MA5-14512), rabbit anti-Ddx21 (1:500, Novus Biologicals NB100-1718S), rabbit anti-p21 (1:2500, Cell Signaling Technology #2947), rabbit anti-p53 (1:1600, Cell Signaling Technology #2527).

Techniques: Comparison, Activity Assay, Fluorescence, Imaging, Immunofluorescence, Staining

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