Incubation:Article Title: Unaffected Li-Fraumeni Syndrome Carrier Parent Demonstrates Allele-Specific mRNA Stabilization of Wild-Type TP53 Compared to Affected Offspring.
Article Snippet: Electroblotted membranes were blocked with 5% non-fat milk (Nestlé Carnation, Solon, OH, USA) in TBS (Cell Signaling Technology, Boston, MA, USA) for 60 min at room temperature. .. They were then incubated with either primary 1:1000 antiphosphoChk1[S345] (Cell Signaling Technology, #2348), 1:1000 anti-Chk1 (Cell Signaling Technology; #2360), 1:1000 anti-phospho-Chk2[T68] (Cell Signaling Technology; #2197), 1:1000 antiChk2 (Cell Signaling Technology; #6334), 1:500 anti-mutant p53 (abcam, Boston, MA, USA; #ab32049), 1:1000 anti-p53 (Santa Cruz Biotechnology, Dallas, TX, USA; #sc-6243), or 1:5000 anti-β-actin (Santa Cruz Biotechnology; #sc-47778) overnight at 4◦C, followed by the corresponding horseradish peroxidase-conjugated goat anti-rabbit (Cell Signaling Technology; #7074) or anti-mouse (Cell Signaling Technology; #7076) secondary IgG for 2 h at room temperature in TBST (Cell Signaling Technology) with 5% non-fat milk (Nestlé Carnation) or 5% BSA (Cell Signaling Technology), according to the antibody manufacturer’s recommendations. ..
Western Blot:Article Title: Adeno-Associated Virus Type 2 Modulates the Host DNA Damage Response Induced by Herpes Simplex Virus 1 during Coinfection
Article Snippet: .. The following primary antibodies were used: anti-actin (Santa Cruz Biotechnology SC-10731; dilution for Western blotting [WB], 1:10,000), anti-ATM (Genetex 70107; dilution for WB, 1:1,000), anti-ATM-P-S1981 (Rockland Immunochemicals 200-301-400; dilution for WB, 1:500; dilution for immunofluorescence [IF] assay, 1:50), antiATR (SC-28901; dilution for WB, 1:1,000), anti-ATR-P-S428 (Cell Signaling Technology CST-2853; dilution for WB, 1:500; dilution for IF assay, 1:200), anti-ATRIP (Abcam Ab-19531; dilution for WB, 1:2,000; dilution for IF assay, 1:500), anti-Chk1 (SC-8408; dilution for WB, 1:1,000), anti-Chk1-P-S345 (CST-133D3; dilution for WB, 1:1,000), antiChk2 (SC-5278; dilution for WB, 1:1,000), anti-Chk2-P-T68 (SC-16297; dilution for WB and immunoprecipitation, 1:250; dilution for IF assay, 1:100), anti-DNA-PKcs (NeoMarkers M5370; dilution for WB, 1:500; dilution for IF assay, 1:50; dilution for flow cytometry, 1:250), anti-DNAPKcs-P-S2056 (Ab-18192; dilution for WB, 1:2,000; dilution for IF assay, 1:500), anti-HSV-1 ICP8 (Ab-20193; dilution for WB, 1:1,000; dilution for IF assay, 1:200), anti-H2AX-P-S139 (Millipore 05-636; dilution for WB, 1:500; dilution for IF assay, 1:50), anti-Nbs1 (Novus Biologicals 100- 143; dilution for WB, 1:1,000), anti-Nbs1-P-S343 (Ab-47272; dilution for WB, 1:500; dilution for IF assay, 1:100), anti-p53 (Ab-1101; dilution for WB, 1:1,000), anti-p53-P-S15 (Ab-38497 [purchased in June 2009 and no longer available]; dilution for WB, 1:500; dilution for IF assay, 1:500), anti-AAV2 Rep (Fitzgerald Industries 10R-A111A; dilution for WB, 1:200), anti-RPA32 (Bethyl Laboratories BL-A300-244A; dilution for WB, 1:2,000; dilution for IF assay, 1:500), anti-RPA32-P-S4/8 (BL-A300-245A; dilution for WB, 1:200; dilution for IF assay, 1:200), and anti-USP7 (CST3277; dilution for WB, 1:750). .. The following secondary antibodies were used: rabbit anti-mouse IgG-horseradish peroxidase (HRP; Sigma A9044; dilution, 1:10,000), goat anti-rabbit IgG-HRP (Sigma A6154; dilution, 1:10,000), rabbit TrueBlot, goat anti-rabbit IgG (H L)-Alexa Fluor 405 (AF405; Molecular Probes A31556; dilution, 1:500), goat anti-rabbit IgG (H L)-AF594 (Molecular Probes A11012; dilution, 1:1,000), goat antimouse IgG (H L)-AF594 (Molecular Probes A11005; dilution, 1:1,000), goat anti-mouse IgG (H L)-fluorescein isothiocyanate (FITC; Southern Biotechnology 1031-02; dilution, 1:200), goat anti-rabbit IgG (H L)FITC (Southern Biotechnology 4050-02; dilution, 1:200), and goat antimouse IgG-Cy5 (Millipore AP181S; dilution, 1:500).
Article Title: The DNA Damage Response Pathway Regulates the Alternative Splicing of the Apoptotic Mediator Bcl-x
Article Snippet: .. Standard protocols were applied for Western blotting using an anti-phospho-H2AX (Upstate and Millipore), anti-ATM (Y170, Abcam), antiCHK2 (a mixture of antibodies from Cell Signaling Technology recognizing phosphorylated Ser19, Ser33/35, Thr68, and Thr432), or anti-p53 antibody (Calbiochem). .. The same blots were decorated with an anti- -actin antibody (A5316, SigmaAldrich) to evaluate total protein content in different lanes.
Immunofluorescence:Article Title: Adeno-Associated Virus Type 2 Modulates the Host DNA Damage Response Induced by Herpes Simplex Virus 1 during Coinfection
Article Snippet: .. The following primary antibodies were used: anti-actin (Santa Cruz Biotechnology SC-10731; dilution for Western blotting [WB], 1:10,000), anti-ATM (Genetex 70107; dilution for WB, 1:1,000), anti-ATM-P-S1981 (Rockland Immunochemicals 200-301-400; dilution for WB, 1:500; dilution for immunofluorescence [IF] assay, 1:50), antiATR (SC-28901; dilution for WB, 1:1,000), anti-ATR-P-S428 (Cell Signaling Technology CST-2853; dilution for WB, 1:500; dilution for IF assay, 1:200), anti-ATRIP (Abcam Ab-19531; dilution for WB, 1:2,000; dilution for IF assay, 1:500), anti-Chk1 (SC-8408; dilution for WB, 1:1,000), anti-Chk1-P-S345 (CST-133D3; dilution for WB, 1:1,000), antiChk2 (SC-5278; dilution for WB, 1:1,000), anti-Chk2-P-T68 (SC-16297; dilution for WB and immunoprecipitation, 1:250; dilution for IF assay, 1:100), anti-DNA-PKcs (NeoMarkers M5370; dilution for WB, 1:500; dilution for IF assay, 1:50; dilution for flow cytometry, 1:250), anti-DNAPKcs-P-S2056 (Ab-18192; dilution for WB, 1:2,000; dilution for IF assay, 1:500), anti-HSV-1 ICP8 (Ab-20193; dilution for WB, 1:1,000; dilution for IF assay, 1:200), anti-H2AX-P-S139 (Millipore 05-636; dilution for WB, 1:500; dilution for IF assay, 1:50), anti-Nbs1 (Novus Biologicals 100- 143; dilution for WB, 1:1,000), anti-Nbs1-P-S343 (Ab-47272; dilution for WB, 1:500; dilution for IF assay, 1:100), anti-p53 (Ab-1101; dilution for WB, 1:1,000), anti-p53-P-S15 (Ab-38497 [purchased in June 2009 and no longer available]; dilution for WB, 1:500; dilution for IF assay, 1:500), anti-AAV2 Rep (Fitzgerald Industries 10R-A111A; dilution for WB, 1:200), anti-RPA32 (Bethyl Laboratories BL-A300-244A; dilution for WB, 1:2,000; dilution for IF assay, 1:500), anti-RPA32-P-S4/8 (BL-A300-245A; dilution for WB, 1:200; dilution for IF assay, 1:200), and anti-USP7 (CST3277; dilution for WB, 1:750). .. The following secondary antibodies were used: rabbit anti-mouse IgG-horseradish peroxidase (HRP; Sigma A9044; dilution, 1:10,000), goat anti-rabbit IgG-HRP (Sigma A6154; dilution, 1:10,000), rabbit TrueBlot, goat anti-rabbit IgG (H L)-Alexa Fluor 405 (AF405; Molecular Probes A31556; dilution, 1:500), goat anti-rabbit IgG (H L)-AF594 (Molecular Probes A11012; dilution, 1:1,000), goat antimouse IgG (H L)-AF594 (Molecular Probes A11005; dilution, 1:1,000), goat anti-mouse IgG (H L)-fluorescein isothiocyanate (FITC; Southern Biotechnology 1031-02; dilution, 1:200), goat anti-rabbit IgG (H L)FITC (Southern Biotechnology 4050-02; dilution, 1:200), and goat antimouse IgG-Cy5 (Millipore AP181S; dilution, 1:500).
Immunoprecipitation:Article Title: Adeno-Associated Virus Type 2 Modulates the Host DNA Damage Response Induced by Herpes Simplex Virus 1 during Coinfection
Article Snippet: .. The following primary antibodies were used: anti-actin (Santa Cruz Biotechnology SC-10731; dilution for Western blotting [WB], 1:10,000), anti-ATM (Genetex 70107; dilution for WB, 1:1,000), anti-ATM-P-S1981 (Rockland Immunochemicals 200-301-400; dilution for WB, 1:500; dilution for immunofluorescence [IF] assay, 1:50), antiATR (SC-28901; dilution for WB, 1:1,000), anti-ATR-P-S428 (Cell Signaling Technology CST-2853; dilution for WB, 1:500; dilution for IF assay, 1:200), anti-ATRIP (Abcam Ab-19531; dilution for WB, 1:2,000; dilution for IF assay, 1:500), anti-Chk1 (SC-8408; dilution for WB, 1:1,000), anti-Chk1-P-S345 (CST-133D3; dilution for WB, 1:1,000), antiChk2 (SC-5278; dilution for WB, 1:1,000), anti-Chk2-P-T68 (SC-16297; dilution for WB and immunoprecipitation, 1:250; dilution for IF assay, 1:100), anti-DNA-PKcs (NeoMarkers M5370; dilution for WB, 1:500; dilution for IF assay, 1:50; dilution for flow cytometry, 1:250), anti-DNAPKcs-P-S2056 (Ab-18192; dilution for WB, 1:2,000; dilution for IF assay, 1:500), anti-HSV-1 ICP8 (Ab-20193; dilution for WB, 1:1,000; dilution for IF assay, 1:200), anti-H2AX-P-S139 (Millipore 05-636; dilution for WB, 1:500; dilution for IF assay, 1:50), anti-Nbs1 (Novus Biologicals 100- 143; dilution for WB, 1:1,000), anti-Nbs1-P-S343 (Ab-47272; dilution for WB, 1:500; dilution for IF assay, 1:100), anti-p53 (Ab-1101; dilution for WB, 1:1,000), anti-p53-P-S15 (Ab-38497 [purchased in June 2009 and no longer available]; dilution for WB, 1:500; dilution for IF assay, 1:500), anti-AAV2 Rep (Fitzgerald Industries 10R-A111A; dilution for WB, 1:200), anti-RPA32 (Bethyl Laboratories BL-A300-244A; dilution for WB, 1:2,000; dilution for IF assay, 1:500), anti-RPA32-P-S4/8 (BL-A300-245A; dilution for WB, 1:200; dilution for IF assay, 1:200), and anti-USP7 (CST3277; dilution for WB, 1:750). .. The following secondary antibodies were used: rabbit anti-mouse IgG-horseradish peroxidase (HRP; Sigma A9044; dilution, 1:10,000), goat anti-rabbit IgG-HRP (Sigma A6154; dilution, 1:10,000), rabbit TrueBlot, goat anti-rabbit IgG (H L)-Alexa Fluor 405 (AF405; Molecular Probes A31556; dilution, 1:500), goat anti-rabbit IgG (H L)-AF594 (Molecular Probes A11012; dilution, 1:1,000), goat antimouse IgG (H L)-AF594 (Molecular Probes A11005; dilution, 1:1,000), goat anti-mouse IgG (H L)-fluorescein isothiocyanate (FITC; Southern Biotechnology 1031-02; dilution, 1:200), goat anti-rabbit IgG (H L)FITC (Southern Biotechnology 4050-02; dilution, 1:200), and goat antimouse IgG-Cy5 (Millipore AP181S; dilution, 1:500).
Flow Cytometry:Article Title: Adeno-Associated Virus Type 2 Modulates the Host DNA Damage Response Induced by Herpes Simplex Virus 1 during Coinfection
Article Snippet: .. The following primary antibodies were used: anti-actin (Santa Cruz Biotechnology SC-10731; dilution for Western blotting [WB], 1:10,000), anti-ATM (Genetex 70107; dilution for WB, 1:1,000), anti-ATM-P-S1981 (Rockland Immunochemicals 200-301-400; dilution for WB, 1:500; dilution for immunofluorescence [IF] assay, 1:50), antiATR (SC-28901; dilution for WB, 1:1,000), anti-ATR-P-S428 (Cell Signaling Technology CST-2853; dilution for WB, 1:500; dilution for IF assay, 1:200), anti-ATRIP (Abcam Ab-19531; dilution for WB, 1:2,000; dilution for IF assay, 1:500), anti-Chk1 (SC-8408; dilution for WB, 1:1,000), anti-Chk1-P-S345 (CST-133D3; dilution for WB, 1:1,000), antiChk2 (SC-5278; dilution for WB, 1:1,000), anti-Chk2-P-T68 (SC-16297; dilution for WB and immunoprecipitation, 1:250; dilution for IF assay, 1:100), anti-DNA-PKcs (NeoMarkers M5370; dilution for WB, 1:500; dilution for IF assay, 1:50; dilution for flow cytometry, 1:250), anti-DNAPKcs-P-S2056 (Ab-18192; dilution for WB, 1:2,000; dilution for IF assay, 1:500), anti-HSV-1 ICP8 (Ab-20193; dilution for WB, 1:1,000; dilution for IF assay, 1:200), anti-H2AX-P-S139 (Millipore 05-636; dilution for WB, 1:500; dilution for IF assay, 1:50), anti-Nbs1 (Novus Biologicals 100- 143; dilution for WB, 1:1,000), anti-Nbs1-P-S343 (Ab-47272; dilution for WB, 1:500; dilution for IF assay, 1:100), anti-p53 (Ab-1101; dilution for WB, 1:1,000), anti-p53-P-S15 (Ab-38497 [purchased in June 2009 and no longer available]; dilution for WB, 1:500; dilution for IF assay, 1:500), anti-AAV2 Rep (Fitzgerald Industries 10R-A111A; dilution for WB, 1:200), anti-RPA32 (Bethyl Laboratories BL-A300-244A; dilution for WB, 1:2,000; dilution for IF assay, 1:500), anti-RPA32-P-S4/8 (BL-A300-245A; dilution for WB, 1:200; dilution for IF assay, 1:200), and anti-USP7 (CST3277; dilution for WB, 1:750). .. The following secondary antibodies were used: rabbit anti-mouse IgG-horseradish peroxidase (HRP; Sigma A9044; dilution, 1:10,000), goat anti-rabbit IgG-HRP (Sigma A6154; dilution, 1:10,000), rabbit TrueBlot, goat anti-rabbit IgG (H L)-Alexa Fluor 405 (AF405; Molecular Probes A31556; dilution, 1:500), goat anti-rabbit IgG (H L)-AF594 (Molecular Probes A11012; dilution, 1:1,000), goat antimouse IgG (H L)-AF594 (Molecular Probes A11005; dilution, 1:1,000), goat anti-mouse IgG (H L)-fluorescein isothiocyanate (FITC; Southern Biotechnology 1031-02; dilution, 1:200), goat anti-rabbit IgG (H L)FITC (Southern Biotechnology 4050-02; dilution, 1:200), and goat antimouse IgG-Cy5 (Millipore AP181S; dilution, 1:500).
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