Journal:

Article Title: Modification of the ionizing radiation response in living cells by an scFv against the DNA-dependent protein kinase

doi: 10.1093/nar/gkg775

Figure Lengend Snippet: (A) Microinjected scFv 18-2 is stable in RPE E1A cells. Cells were fixed at 6 h after injection and stained with DAPI, 1:1000 anti-DNA-PKcs human autoimmune serum and 1:250 anti-E tag mouse monoclonal antibody to detect scFv18-2. (a–e) Non-injected cell. (f–j) Microinjected cell showing nuclear pattern of scFv distribution. (k–o) Microinjected cell showing cytoplasmic pattern of scFv distribution. (B) A combination of microinjected scFv 18-2 and irradiation blocks colony formation. (a–e) scFv 18-2 was co-injected with pEGFP N1 vector into RPE E1A cells. Living cells were observed after 16 h with a combination of phase contrast and epifluorescence illumination. Green fluorescence indicates EGFP expression. (f–j) Cells were fixed after 88 h and stained with 1:500 anti-GFP antibody (Novus Biologicals, Littleton, CO) and 1:500 Alexa-Fluor 488-conjugated goat anti-rabbit antibody (Molecular Probes, Eugene, OR). The same field as in (a–e) was observed with DIC and fluorescence. (C) A combination of microinjected scFv 18-2 and irradiation induces apoptosis. scFv 18-2-injected RPE E1A cells were fixed at 60 h after injection and stained with DAPI (blue), anti-GFP antibody (green) and anti-active caspase 3 antibody (red). Scale bar indicates 10 µm in all panels.

Article Snippet: Samples were incubated with one or more of the following antibodies: 1:250 dilution of anti-E-tag (detect scFv; Amersham Pharmacia Biotech, Piscataway NJ), 1:1000 anti-DNA-PKcs (human serum FT) ( 31 ), 1:500 anti-green fluorescent protein (Novus Biologicals, Littleton, CO or Molecular Probes, Eugene, OR), 1:200 anti-activated caspase 3 (Promega) or 1:500 anti-γ-H2AX (mAb JBW301; Upstate Cell Signaling Solutions, Waltham, MA).

Techniques: Injection, Staining, Irradiation, Plasmid Preparation, Fluorescence, Expressing

Journal:

Article Title: Modification of the ionizing radiation response in living cells by an scFv against the DNA-dependent protein kinase

doi: 10.1093/nar/gkg775

Figure Lengend Snippet: scFv 18-2 prolongs the lifetime of γ-H2AX foci. (A) SK-MEL-28 cells were injected with scFv 18-2 or with scFv 147 and irradiated at 1.5 Gy. Cells were fixed at 30 or 90 min after irradiation and stained with DAPI (blue), anti-γ-H2AX antibody (red) and anti-GFP antibody as described in Materials and Methods. (B) As in (A), except with 0.1 Gy of ionizing radiation. Scale bar indicates 10 µm in both panels. There were 50–100 successfully injected cells in each experimental group, and results shown are typical.

Article Snippet: Samples were incubated with one or more of the following antibodies: 1:250 dilution of anti-E-tag (detect scFv; Amersham Pharmacia Biotech, Piscataway NJ), 1:1000 anti-DNA-PKcs (human serum FT) ( 31 ), 1:500 anti-green fluorescent protein (Novus Biologicals, Littleton, CO or Molecular Probes, Eugene, OR), 1:200 anti-activated caspase 3 (Promega) or 1:500 anti-γ-H2AX (mAb JBW301; Upstate Cell Signaling Solutions, Waltham, MA).

Techniques: Injection, Irradiation, Staining