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Image Search Results
Journal: PLoS Pathogens
Article Title: Parvovirus B19 NS1 protein induces cell cycle arrest at G2-phase by activating the ATR-CDC25C-CDK1 pathway
doi: 10.1371/journal.ppat.1006266
Figure Lengend Snippet: NS1-S1 and NS1 mTAD2 -S1 cells were treated with Dox (5 μg/ml) and collected and lysed 72 h later. (A) Cell lysates were analyzed for expression of cyclin B1, phosphorylated CDK1 (CDK1(pY15) and CDK1(pT161), lower band ), and total CDK1 by Western blotting. (B) Cell lysates were analyzed for expression of ATM(pS1981), ATM, CHK2, CHK2(pS33/35), and β-actin by Western blotting. (C) Cell lysates were analyzed for expression of p21 by Western blotting with an anti-p21 antibody. β-actin was used as a loading control. Untreated UT7/Epo-S1 (S1) cells, S1 cells treated with nocodazole (Noco), and S1 cells treated with hydroxyurea (HU) were used as controls.
Article Snippet: The following first antibodies were purchased: anti-Strep (A01732) from GenScript, Piscataway, NJ; mouse anti-BrdU (clone B44) (347580),
Techniques: Expressing, Western Blot
Journal: PLoS Pathogens
Article Title: Parvovirus B19 NS1 protein induces cell cycle arrest at G2-phase by activating the ATR-CDC25C-CDK1 pathway
doi: 10.1371/journal.ppat.1006266
Figure Lengend Snippet: (A) Western blot analysis. NS1-S1 and NS1 mTAD2 -S1 cells were treated with Dox for 72 h, collected, and lysed, and the nuclei were extracted. Western blot analysis was then performed to detect cyclin B1 and phosphorylated CDK1(pY15), as well as nuclear histone H3 and cytoplasmic GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase). Nuclear extracts from S1 cells and S1 cells treated with nocodazole (Noco) were loaded as controls. (B&C) In vitro CDK1 kinase assay. Equivalent amounts of proteins derived from whole cell lysate (B) or nuclear extracts (C) from S1 cells, S1 cells treated with Noco, and Dox-induced NS1-S1 and NS1 mTAD2 -S1 cells were immunoprecipitated with anti-cyclin B1-crosslinked protein A/G Plus agarose beads for in vitro CDK1 kinase assay. The final products were resolved on a 12% SDS-polyacrylamide gel. The gel was then dried prior to autoradiography, and the phosphorylated histone H1 (pHistone H1) is indicated. (C) Lower panel: Co-Immunoprecipitation (Co-IP) of cyclin B1 and CDK1(pY15) in the nuclear extracts. Nuclear extracts prepared from S1, S1/Noco, NS1-S1/Dox+, and NS1 mTAD2 -S1/Dox+ cells were immunoprecipitated with an anti-cyclin B1 antibody. The eluted proteins were analyzed for CDK1(pY15) by Western blotting. Normal mouse IgG (IgG Ctrl) was used as negative control of immunoprecipitation of control S1 cell extracts.
Article Snippet: The following first antibodies were purchased: anti-Strep (A01732) from GenScript, Piscataway, NJ; mouse anti-BrdU (clone B44) (347580),
Techniques: Western Blot, In Vitro, Kinase Assay, Derivative Assay, Immunoprecipitation, Autoradiography, Co-Immunoprecipitation Assay, Negative Control
Journal: PLoS Pathogens
Article Title: Parvovirus B19 NS1 protein induces cell cycle arrest at G2-phase by activating the ATR-CDC25C-CDK1 pathway
doi: 10.1371/journal.ppat.1006266
Figure Lengend Snippet: NS1 activates ATR through its TAD domain. Activated ATR then transduces signals to CDC25C through activating CHK1. CDC25C phosphatase activity is negatively regulated by phosphorylation at serine 216, which then creates a binding site for the 14-3-3 protein in the cytoplasm . Thus, inactive CDC25C cannot dephosphorylate the cyclin B1/CDK1(pT14/Y15) complex; the latter component is inactive, and so progression from G2- to M-phase is blocked. It is proposed that DNA replication-induced DDR, and thereafter ATR/CHK1 activation, which plays a role in viral DNA replication, should not be involved in CDC25C phosphorylation (see the section for further explanation). The question mark indicates potential modification of CHK1, in addition to the phosphorylation at S345.
Article Snippet: The following first antibodies were purchased: anti-Strep (A01732) from GenScript, Piscataway, NJ; mouse anti-BrdU (clone B44) (347580),
Techniques: Activity Assay, Binding Assay, Activation Assay, Modification