rabbit anti human ddx21  (Novus Biologicals)

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: Breast Cancer Research : BCR

Article Title: Elevated DDX21 regulates c-Jun activity and rRNA processing in human breast cancers

doi: 10.1186/s13058-014-0449-z

Figure Lengend Snippet: DDX21 is overexpressed in breast carcinoma tissues. (A) Immunofluorescence was performed on a human tissue array including 67 cases of malignant breast cancer tissue specimen. Red color depicts DDX21 expression. Blue color depicts DAPI-stained nuclei. Typical images of benign and malignant breast cancer tissues are shown. Left images were cropped from a larger field, while rights images were cropped from a smaller field. All images were taken at 40X magnification. (B) Immunohistochemistry was performed on a human tissue array including 50 cases of invasive ductal carcinoma with adjacent normal tissue samples. Brown color indicated positive DDX21 expression tissues. (C) Immunohistochemistry was performed on a human tissue array including 70 cases of various stages of breast carcinoma. Typical images of positive-stained samples are shown. DAPI, 4',6-diamidino-2-phenylindole.

Article Snippet: A rabbit polyclonal anti-human DDX21 antibody (Bethyl Laboratories) was used at 1:25.

Techniques: Immunofluorescence, Expressing, Staining, Immunohistochemistry

Journal: Breast Cancer Research : BCR

Article Title: Elevated DDX21 regulates c-Jun activity and rRNA processing in human breast cancers

doi: 10.1186/s13058-014-0449-z

Figure Lengend Snippet: DDX21 is highly expressed in proliferative breast cancer cell lines and is both nuclear and nucleolar. (A) A panel of 13 different breast cancer cell lines and nontransformed MCF10A cells were analyzed for DDX21 protein expression by western blot analysis. A total of 50 μg of whole cell extracts was used for analysis. Tubulin was used as a loading control. Quantitation of the signal was made by Image J software and normalized by tubulin levels. (B) Equal numbers of the indicated breast cancer cell lines were plated and cell numbers were counted on a daily basis. Bars indicate standard deviation from three separate experiments. Doubling times were estimated based on the growth curves. Doubling time of MDA-MB-231 cells was determined separately. (C) MCF10A cells were cultured in regular DMEM media without insulin and EGF supplements for 48 hours. Cells were then fed with DMEM complete media with either insulin alone, EGF alone or both insulin and EGF together for 24 hours. Cells were then harvested and total cell lysates were subjected to western blot analysis with anti-DDX21 antibody and anti-tubulin antibody. (D) Above-mentioned growth factor-starved MCF10A cells were treated with 100 ng/ml EGF containing complete media for the indicated time points. Whole cell extracts were subjected to western blot analysis with anti-DDX21 antibody and anti-tubulin antibody. Standard error of the mean is indicated with fold-change. * , P <0.05. (E) HMECs and breast cancer cell lines were subjected to immunofluorescence analysis with anti-DDX21 to detect cellular localization of DDX21 protein (red). Cell nuclei were demarcated by DAPI (blue) and nucleoli were demarcated by UBF (green). DAPI, 4',6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; EGF, epithelial growth factor; HMEC, human mammary epithelial cell; UBF, upstream binding factor.

Article Snippet: A rabbit polyclonal anti-human DDX21 antibody (Bethyl Laboratories) was used at 1:25.

Techniques: Expressing, Western Blot, Control, Quantitation Assay, Software, Standard Deviation, Cell Culture, Immunofluorescence, Modification, Binding Assay

Journal: Breast Cancer Research : BCR

Article Title: Elevated DDX21 regulates c-Jun activity and rRNA processing in human breast cancers

doi: 10.1186/s13058-014-0449-z

Figure Lengend Snippet: DDX21 knockdown in multiple breast cancer cell lines induces cell death and cell cycle arrest. (A) MCF-7 and T47D breast cancer cells were either infected with shSCR or shRNA-DDX21 lentiviruses. Three days postinfection, cells were split and cultured for 48 hours. Typical images for the live culture of the indicated transduced cells are shown. (B) MDA-MB-231 cells or HCC1806 cells were either transduced with shSCR or shRNA-DDX21 lentiviruses. Whole cell lysates were subjected to western blot analysis four days and five days postinfection with anti-DDX21 and anti-tubulin antibodies. (C) MDA-MB-231 cells were either transduced with shSCR or shRNA-DDX21 lentiviruses. Five days postinfection, cells were then subjected to apoptosis analysis by flow cytometry with Vybrant apoptosis kit after PI labeling of nuclei and FITC labeling of Annexin V. Percentage of live and dead cells are shown in each quadrant. (D) Quantitation of live and apoptotic cells were calculated and graphed. (E) Above-mentioned MDA-MB-231 cells were subjected to fixation and PI staining before flow cytometry to detect cell cycle distribution. Percentage of cells in each cell cycle phase is presented. (F) HCC1806 cells were either transduced with shSCR or shRNA-DDX21 lentiviruses. Five days postinfection, cells were subjected to apoptosis analysis. Population of live and dead cells is marked in each quadrant. (G) Quantitation of the live and apoptotic cells is graphed. FITC, fluorescein isothiocyanate.

Article Snippet: A rabbit polyclonal anti-human DDX21 antibody (Bethyl Laboratories) was used at 1:25.

Techniques: Knockdown, Infection, shRNA, Cell Culture, Transduction, Western Blot, Flow Cytometry, Labeling, Quantitation Assay, Staining

Journal: Breast Cancer Research : BCR

Article Title: Elevated DDX21 regulates c-Jun activity and rRNA processing in human breast cancers

doi: 10.1186/s13058-014-0449-z

Figure Lengend Snippet: DDX21 is important for the tumorigenicity of breast cancer cells in vitro and in vivo . (A) T47D, MCF-7 and SKBR3 cells were transduced with either shSCR or shRNA-DDX21 lentiviruses. DDX21 levels were detected by western blot analysis after lentiviral knockdown. Tubulin was used as a loading control. (B) Above-mentioned cells were also subjected to soft agar analysis after plating 1 × 10 4 cells in each 60 mm dish. Typical image is shown after culturing for two weeks. (C) Quantitation of colony numbers in the soft agar assay is graphed; error bars were taken from triplicates. (D) A group of five SCID mice were injected at the mammary gland on both sides with MDA-MB-231-luciferase reporter cells transduced with either shScrambled or with shDDX21 (50,000 cells per injection). Mice were allowed to recover from the surgery and bioluminescence imaging was performed from two weeks to nine weeks. A representative mouse from each group is shown at the indicated time points. Data presented show the mean × standard deviation from five different mice in each group. SCID, severe combined immunodeficiency.

Article Snippet: A rabbit polyclonal anti-human DDX21 antibody (Bethyl Laboratories) was used at 1:25.

Techniques: In Vitro, In Vivo, Transduction, shRNA, Western Blot, Knockdown, Control, Quantitation Assay, Soft Agar Assay, Injection, Luciferase, Imaging, Standard Deviation

Journal: Breast Cancer Research : BCR

Article Title: Elevated DDX21 regulates c-Jun activity and rRNA processing in human breast cancers

doi: 10.1186/s13058-014-0449-z

Figure Lengend Snippet: c-Jun interacts with DDX21 in breast cancer cell lines. (A) Panel of breast cancer cell lines was screened by western blot analysis with anti-c-Jun antibody. Fifty μg of whole cell lysates were subjected to the analysis and tubulin is used as a loading control. Quantitation of the signal was made by Image J software and normalized by tubulin levels. (B) 500 μg of protein lysates were subjected to immunoprecipitation with anti-c-Jun antibody with mouse IgG as a negative control, 25 μg of SKBR3 cell lysate was used as input control. Immune complexes were blotted with anti-DDX21 and anti-c-Jun. (C) Reciprocal immunoprecipitation was also performed with the above-mentioned cell lysates. Western blot analysis was performed to confirm the association between c-Jun and DDX21 in breast cancer cell lines. (D) c-Jun immunoprecipitation was performed on purified nuclear lysates from the indicated cell lines. Western blot analysis was performed to confirm the association between c-Jun and DDX21.

Article Snippet: A rabbit polyclonal anti-human DDX21 antibody (Bethyl Laboratories) was used at 1:25.

Techniques: Western Blot, Control, Quantitation Assay, Software, Immunoprecipitation, Negative Control, Purification

Journal: Breast Cancer Research : BCR

Article Title: Elevated DDX21 regulates c-Jun activity and rRNA processing in human breast cancers

doi: 10.1186/s13058-014-0449-z

Figure Lengend Snippet: DDX21 modulates AP-1 activity in MDA-MB-231 cells. (A) MDA-MB-231 cells were either infected with shSCR or shRNA-DDX21 lentiviruses. Whole cell lysates were subjected to western blot analysis with the indicated antibodies with tubulin as an internal control. Standard error of the mean is indicated with fold-change. * , P <0.05. (B) RT-PCR analysis was performed for cyclin D1 mRNA levels after normalization with GAPDH mRNA levels. Error bars were taken from three independent experiments. * , P <0.001. (C) RT-PCR analysis was performed for EGFR mRNA levels after normalization with GAPDH mRNA levels. Error bars were taken from three independent experiments. * , P <0.005. (D) MDA-MB-231 and SKBR3 cells were infected with AP-1 reporter lentiviruses to detect endogenous AP-1 activity. After verification of AP-1 luciferase activity, these cells were infected with either shSCR or shRNA-DDX21. Two days postinfection, equal numbers of cells were transfected with pGL- Renilla- luciferase plasmids. Equal numbers of cells were then analyzed for both firefly and Renilla luciferase activity. Data presented is firefly luciferase activity after normalization with Renilla luciferase and then further normalized to shSCR control. * , P <0.001 (n =3). (E) MDA-MB-231 cells infected with shSCR or shRNA-DDX21 lentiviruses were stained with antibodies recognizing NPM and visualized by indirect immunofluorescence. DAPI stain was used to mark nuclei. Images are representative of over 100 cells for each condition. DAPI, 4',6-diamidino-2-phenylindole; EGFR, epithelial growth factor receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NPM, nucleophosmin; RT-PCR, reverse transcriptase-polymerase chain reaction.

Article Snippet: A rabbit polyclonal anti-human DDX21 antibody (Bethyl Laboratories) was used at 1:25.

Techniques: Activity Assay, Infection, shRNA, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Luciferase, Transfection, Staining, Immunofluorescence, Reverse Transcription, Polymerase Chain Reaction

Journal: Breast Cancer Research : BCR

Article Title: Elevated DDX21 regulates c-Jun activity and rRNA processing in human breast cancers

doi: 10.1186/s13058-014-0449-z

Figure Lengend Snippet: Nucleolar DDX21 promotes rRNA processing in breast cancer cells. (A) HCC1806 cells were infected with shSCR or shRNA-DDX21, two days postinfection, cells were pulsed with [ 3 H-methyl] methionine and chased for 1 hour. Total RNA was extracted and analyzed for 47S, 32S, 28S and 18S rRNA levels. Methylene blue-stained membrane is also shown at the bottom. (B) HCC1806 cells were infected with shSCR or shRNA-DDX21, two days postinfection, cells were pulsed with [ 3 H-methyl] methionine and chased for 0, 25 and 50 min. Total RNA was extracted and analyzed for 47S, 32S, 28S and 18S rRNA levels. Methylene blue-stained membrane is also shown at the bottom.

Article Snippet: A rabbit polyclonal anti-human DDX21 antibody (Bethyl Laboratories) was used at 1:25.

Techniques: Infection, shRNA, Staining, Membrane

Journal: Breast Cancer Research : BCR

Article Title: Elevated DDX21 regulates c-Jun activity and rRNA processing in human breast cancers

doi: 10.1186/s13058-014-0449-z

Figure Lengend Snippet: DDX21 helicase activity is required for enhanced Ras V12 transformation. (A) Arf -null MEFs were infected with lentiviruses that encode either wild-type human DDX21 or K236R mutant DDX21 with empty vector as a control. Infected cells were then selected in hygromycin-containing media (500 μg/ml) for three days. Cells were then plated and infected with retrovirus encoding Ras V12 or empty vector, and selected in puromycin (4 μg/ml) for two days. Cells were then infected with lentiviruses targeting mouse endogenous DDX21 (shRNA-DDX21) with shSCR as a control. Four days postinfection, whole cell lysate for each sample was then analyzed by western blot with anti-DDX21 antibody, anti-tubulin antibody and anti-Ras antibody. (B) For above-mentioned cells, 1 × 10 4 cells were plated into soft agar for colony-forming assay. Quantitation for the number of colonies was shown. Data presented is the average number of colonies from triplicate plates. Bars indicate standard deviation from each triplicate. * , P <0.001. (C) Above-mentioned cells (1 × 10 6 per condition) were injected into the flanks of NUDE mice, after four weeks, mice were sacrificed and tumor were dissected and photographed. (D) Tumors from each group were weighed and graphed. (E) Above-mentioned cells were fixed and stained with antibodies recognizing DDX21 and visualized by indirect immunofluorescence. DAPI was used to mark nuclei. Images are representative of over 100 cells counted for each condition. DAPI, 4',6-diamidino-2-phenylindole; MEFs, mouse embryonic fibroblasts.

Article Snippet: A rabbit polyclonal anti-human DDX21 antibody (Bethyl Laboratories) was used at 1:25.

Techniques: Activity Assay, Transformation Assay, Infection, Mutagenesis, Plasmid Preparation, Control, shRNA, Western Blot, Quantitation Assay, Standard Deviation, Injection, Staining, Immunofluorescence