Journal: PLoS ONE

Article Title: DNA-PK Target Identification Reveals Novel Links between DNA Repair Signaling and Cytoskeletal Regulation

doi: 10.1371/journal.pone.0080313

Figure Lengend Snippet: (A) Double immunostaining of anti-γ-H2AX (red) and anti-vimentin (S459-P) (green) in MRC-5 cells transfected with Dbait32Hc or 8H or irradiated (10Gy). The arrow indicates a cell negative for both γ-H2AX and phosphorylated vimentin (S459). (B) MRC-5, SK28, M059K or DNA-PK-deficient M059J cells were transfected with Dbait32Hc or 8H or irradiated (IR) with 10 Gy, as indicated. Proteins were extracted 1 h after the end of treatment for western blot analysis and the lysates were probed for vimentin (Vim-S459-P), γ-H2AX and β-actin. (C) MRC-5 cells were treated 2 h before and during Dbait32Hc or 8H treatment with 10 µM Nu7026 (DNA-PK inhibitor), 20 µM wortmannin (PIKK inhibitor), 10 µM KU-55933 (ATM inhibitor) or vehicle (DMSO). The total cell extracts were processed and probed as in (B). (D) The indicated cell lines were transfected with Dbait32Hc or 8H, fixed 1 h after the end of transfection and immunostained for vimentin (Vim-S459-P) (green) and γ-H2AX (red). (E) Double immunostaining of 32Hc- or 8H-treated SK28 cells with antibodies against total vimentin (Vim, red) and phosphorylated vimentin (Vim-S459-P, green). DNA was stained with DAPI (blue). Scale bar: 20 µm.

Article Snippet: The rabbit polyclonal anti-vimentin-S459-P antibody was produced and purified by BioGenes (Berlin, Germany) and was directed against the C-INET(pS)QHHDD peptide (BioGenes).

Techniques: Double Immunostaining, Transfection, Irradiation, Western Blot, Staining

Journal: PLoS ONE

Article Title: DNA-PK Target Identification Reveals Novel Links between DNA Repair Signaling and Cytoskeletal Regulation

doi: 10.1371/journal.pone.0080313

Figure Lengend Snippet: (A) Merged images of immunostaining for vimentin (S459-P) (green) and DNA-staining with DAPI (blue) in untreated MRC-5 cells at the indicated stages of mitosis. (B) Immunostaining as in (A) of MO59K (DNA-PK +/+ ) or DNA-PK-deficient M059J (DNA-PK -/- ) cells and ATM-defective AT5BI cells. (C) Immunostaining as in (A) of MRC-5 cells treated for 5 h with 10 µM NU7026 (DNA-PK inhibitor), 20 µM wortmannin (PIKK and PI3K inhibitor), 10 µM KU-55933 (ATM inhibitor) or (D) 60 µM roscovitine (CDK inhibitor). (E) MOS depletion does not affect vimentin phosphorylation at Ser459 during mitosis. HeLa cells were transfected with a control (CTL) or Mos-silencing siRNAs for 72h, followed by fixation and immunostaining as in A. (A-E) Cells in mitosis (arrow) or interphase are shown. Scale bar: 20 µm. (F) MOS depletion verification. HeLa cells were transfected with a control (CTL) or Mos-silencing siRNAs for 72h, followed by protein extraction, which were subjected to western blotting and the blots were probed with antibodies against MOS and β-actin. (G) MOS expression level in MRC-5 cells and HeLa cells. (H) MOS depletion does not affect Dbait32Hc-induced vimentin phosphorylation on Ser459. HeLa cells were transfected as described previously (E), 72h after transfection cells were treated with Dbait32Hc or 8H followed by proteins extraction, then subjected to western blotting and the blots were probed with vimentin (Vim-S459-P), γ-H2AX and β-actin.

Article Snippet: The rabbit polyclonal anti-vimentin-S459-P antibody was produced and purified by BioGenes (Berlin, Germany) and was directed against the C-INET(pS)QHHDD peptide (BioGenes).

Techniques: Immunostaining, Staining, Transfection, Protein Extraction, Western Blot, Expressing

Journal: PLoS ONE

Article Title: DNA-PK Target Identification Reveals Novel Links between DNA Repair Signaling and Cytoskeletal Regulation

doi: 10.1371/journal.pone.0080313

Figure Lengend Snippet: (A) SK-28 cells were transfected with Dbait32Hc or control 8H, fixed 1 h after the end of transfection and immunostained for vimentin (red) and phospho-vimentin (S459-P) (green). A higher magnification of typical cell protrusions is shown. The bar chart provides a quantitative representation of the signal intensity ratio of vimentin (S459-P) and total vimentin for cell protrusions and cytoplasm, with standard deviations, from three analyses of 50 cells per set of conditions. DNA was stained with DAPI (blue). Scale bar: 20 µm. (B) The indicated cell lines were transfected with Dbait32Hc or 8H, wounded 1 h after the end of transfection and wound healing was observed by videomicroscopy for 16 h. The bar chart gives the percentage wound closure, with standard deviations, from three independent experiments. (C) The indicated cell lines were transfected with Dbait32Hc or 8H, then 1 h after the end of transfection cells were detached by trypsinization and let to adhere on fibronectin for 1h. The bar chart gives the relative adhesion to fibronectin measure by colorimetric assay, with standard deviations, from two independent experiments (Ab560). The significance of differences was assessed by the Kruskal-Wallis method. *, p <0.0005.

Article Snippet: The rabbit polyclonal anti-vimentin-S459-P antibody was produced and purified by BioGenes (Berlin, Germany) and was directed against the C-INET(pS)QHHDD peptide (BioGenes).

Techniques: Transfection, Staining, Colorimetric Assay

Journal: PLoS ONE

Article Title: Cellular Expression Profile for Interstitial Cells of Cajal in Bladder - A Cell Often Misidentified as Myocyte or Myofibroblast

doi: 10.1371/journal.pone.0048897

Figure Lengend Snippet: Cryosections of mouse bladders were labeled with antibodies to vimentin (A. green), desmin (D. green), PDGFβ receptor (G. green), and NTPDase2 (B. E. H. red) and Topro-3 to label nuclei (B. blue). Color merged panels are shown on the right (C. F. I). Merged signals of NTPDase2 and vimentin, desmin, and PDGFβ receptor are shown as yellow (C. F. I). White arrows indicate representative co-localization. White asterisks indicate non-co-localized signal of smooth muscle (I) and fibroblasts (F. I). White scale bars = 10 µm.

Article Snippet: Affinity-purified polyclonal rabbit anti-c-kit antibody (A0501), affinity-purified polyclonal rabbit anti-connexin 43 antibody (C0158), affinity-purified polyclonal rabbit anti-vimentin antibody (C0390), affinity-purified polyclonal rabbit anti-desmin antibody (C0171), affinity-purified polyclonal rabbit anti-PDGFβ antibody (B7194), affinity-purified polyclonal rabbit anti-merlin antibody (A8046) were purchased from Assay Biotechnology (Sunnyvale, CA).

Techniques: Labeling

Journal: PLoS ONE

Article Title: Cellular Expression Profile for Interstitial Cells of Cajal in Bladder - A Cell Often Misidentified as Myocyte or Myofibroblast

doi: 10.1371/journal.pone.0048897

Figure Lengend Snippet: Summary of expression profile of molecular markers in bladder ICC.

Article Snippet: Affinity-purified polyclonal rabbit anti-c-kit antibody (A0501), affinity-purified polyclonal rabbit anti-connexin 43 antibody (C0158), affinity-purified polyclonal rabbit anti-vimentin antibody (C0390), affinity-purified polyclonal rabbit anti-desmin antibody (C0171), affinity-purified polyclonal rabbit anti-PDGFβ antibody (B7194), affinity-purified polyclonal rabbit anti-merlin antibody (A8046) were purchased from Assay Biotechnology (Sunnyvale, CA).

Techniques: Expressing