Journal:

Article Title: A Rad50-dependent pathway of DNA repair is deficient in Fanconi anemia fibroblasts

doi: 10.1093/nar/gkh649

Figure Lengend Snippet: Effect of introduction of anti-Rad50, anti-Mre11 or anti-Nbs1 antibodies on DNA end-joining frequency in normal and FA fibroblasts. (A) The rejoining frequency of cohesive-ended DNA was determined in the HDFs in the absence of antibody (white bars) or in the presence of anti-Rad50 antibody (black bars). HDF, normal HDFs; FA-A, FA fibroblasts belonging to complementation group A; FA-C, FA fibroblasts belonging to complementation group C; FA-G, FA fibroblasts belonging to complementation group G. *P < 0.0001 compared to the same cell strain in the absence of antibody, χ2-test. (B) The rejoining frequency of blunt-ended DNA was determined in the same cells as in (A). *P < 0.0001 compared to the same cell type in the absence of antibody, χ2-test. (C) The rejoining frequency of cohesive- and blunt-ended DNA was determined in normal diploid fibroblasts co-electroporated with non-specific IgG (1), anti-Rad50 antibody (2), anti-Mre11 antibody (3), anti-Nbs1 antibody (4), both anti-Rad50 and anti-Mre11 antibodies (5), both anti-Rad50 and anti-Nbs1 antibodies (6), both anti-Mre11 and anti-Nbs1 antibodies (7), and anti-Rad50, anti-Mre11 and anti-Nbs1 antibodies (8). In all cases, antibody treatment significantly reduced plasmid end-joining levels compared to those observed in cells not treated with antibody, P < 0.0001, χ2-test.

Article Snippet: Mouse polyclonal anti-DNA ligase III, rabbit polyclonal anti-Fancd2, rabbit polyclonal anti-Rad50, rabbit polyclonal anti-Mre11 and rabbit polyclonal anti-Nbs1 antibodies were obtained from Novus Biologicals, Inc. (Littleton, CO).

Techniques: Plasmid Preparation

Journal:

Article Title: A Rad50-dependent pathway of DNA repair is deficient in Fanconi anemia fibroblasts

doi: 10.1093/nar/gkh649

Figure Lengend Snippet: Introduction of antibodies directed against proteins in the RMN complex sensitizes normal diploid fibroblasts to agents that damage chromosomal DNA. (A) Cell death was determined following treatment with 20 U of the restriction endonuclease PvuII in cells co-electroporated with non-specific IgG (1), anti-Rad50 antibody (2), anti-Mre11 antibody (3), anti-Nbs1 antibody (4), both anti-Rad50 and anti-Mre11 antibodies (5), both anti-Rad50 and anti-Nbs1 antibodies (6), both anti-Mre11 and anti-Nbs1 antibodies (7), and anti-Rad50, anti-Mre11 and anti-Nbs1 antibodies (8). (B) Cell death was determined after treatment with 200 ng/ml diepoxybutane of cells electroporated with the same antibodies as in (A).

Article Snippet: Mouse polyclonal anti-DNA ligase III, rabbit polyclonal anti-Fancd2, rabbit polyclonal anti-Rad50, rabbit polyclonal anti-Mre11 and rabbit polyclonal anti-Nbs1 antibodies were obtained from Novus Biologicals, Inc. (Littleton, CO).

Techniques:

Journal: Oncotarget

Article Title: Overexpression of karyopherin-α2 in cholangiocarcinoma correlates with poor prognosis and gemcitabine sensitivity via nuclear translocation of DNA repair proteins

doi: 10.18632/oncotarget.15020

Figure Lengend Snippet: A . TFK1 cells were transfected with KPNA2-specific siRNA, and the protein expression of KPNA2 and the MRN complex in both the nucleus and cytoplasm was analyzed using Western blotting. β-actin was used as a loading control. B . Intracellular localization of KPNA2 and the MRN complex in TFK1 cells treated with KPNA2 or control siRNA. TFK1 cells were immunostained with antibodies against KPNA2 (green) and MRE11, RAD50, and NBS1 (red). All sections were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI, blue). Original magnification: ×600; scale bar, 20 μm. C . Intracellular localization of KPNA2 and the MRN complex in clinical cholangiocarcinoma tissue. Representative cholangiocarcinoma tissue was immunostained with antibodies against KPNA2 (green) and MRE11, RAD50, and NBS1 (red). All sections were counterstained with DAPI (blue). Original magnification: ×400; scale bar, 100 μm.

Article Snippet: The sections were then incubated with rabbit anti-KPNA2 polyclonal antibody (1:400; ab84440, anti-KPNA2 antibody, Abcam, Cambridge, England), anti-MRE11 rabbit monoclonal antibody (1:400; ab109623, anti-Mre11 antibody, Abcam), anti-RAD50 mouse monoclonal antibody (1:400; ab89, anti-Rad50 antibody, Abcam), or anti-p95 NBS1 rabbit monoclonal antibody (1:400; ab32074, anti-p95 NBS1 antibody, Abcam) in Can Get Signal ® immunostain (Toyobo Co., Ltd., Osaka, Japan) overnight at 4°C and at room temperature for 30 min.

Techniques: Transfection, Expressing, Western Blot

Journal: Journal of Virology

Article Title: The Papillomavirus E1 Helicase Activates a Cellular DNA Damage Response in Viral Replication Foci

doi: 10.1128/jvi.00541-11

Figure Lengend Snippet: FIG. 2. HPV16 E1 activates the pChk1, pChk2, and H2AX proteins. CV-1 cells were transfected with an HPV16 E1 expression plasmid or empty vectors (pMEP9) on coverslips. Cells were stained and with chicken anti-EE (-E1) (in green) and antibodies to various DNA damage response (DDR) proteins, pChk1 (Ser345), pChk2 (Thr68), and histone H2AX (Ser139) (in red).

Article Snippet: The following antibodies and dilutions were used for immunofluorescence: chicken anti-EE (Bethyl Laboratories, Inc., A190-109A; 1:100), M2 mouse and rabbit anti-Flag (Sigma F3165 and F7425; 1:500 and 1:200, respectively), rabbit antiphosphorylated Thr68 Chk2 (Cell Signaling 2661; 1:100), mouse anti-phosphor- on N ovem ber 18, 2012 by U N IV E R S IT Y O F A R IZ O N A LIB R A R Y http://jvi.asm .org/ D ow nloaded from ylated Ser1981 ATM (Abcam ab81292; 1:100), mouse anti-phosphorylated Ser15 p53 (Cell Signaling 9286; 1:100), rabbit anti-phosphorylated Ser139 H2AX (Cell Signaling 2577; 1:100), rabbit anti-phosphorylated Ser345 pChk1 (Cell Signaling 2349; 1:100), rabbit anti-NBS1 (Novus NB100-143; 1:200), and rabbit anticaspase-3 cleaved at Asp175 (Cell Signaling 9661; 1:200).

Techniques: Transfection, Expressing, Plasmid Preparation, Staining