Anti γh2ax phosphor ser 139 sc 517

Indirect immunofluorescence assay (IFA) was performed to evaluate γH2AX foci formation. RKO cells were grown on slides, pre-treated or not with ceapinA7 and then treated with DPE (100 μM) or Thapsigargin (30 nM). After 48 h of treatments, cells were processed as previously described [33 (link)]. Thereafter, cells were incubated with mouse primary monoclonal antibody anti-γH2AX (phosphor-Ser 139) (sc-517; Santa Cruz Biotechnology) for 1 h at room temperature. Subsequently, cells were incubated with a polyclonal conjugated-Cy3 sheep anti-mouse antibody (Jackson ImmunoResearch, Cambridge, UK) for 30 min at room temperature and stained with DAPI (Sigma-Aldrich) for 1 min at room temperature. Slides were analyzed with Apotome Axio Observer Z1 inverted microscope (Zeis, Wetzlar, Germany) equipped with an AxioCam MRM Rev.3 at ×40 magnification. Foci number analysis was performed by Image J software (1.47 version, NIH, Bethesda, MD, USA).

Indirect immunofluorescence assay (IFA) was performed to evaluate γ-H2AX foci formation. Akata cells were plated in 6-well plates as reported above, and were pre-treated with IRE1 RNAse inhibitor 4μ8C (15 μM) for 1 h, then treated with AZD2461 (10 μM), which is a PARP inhibitor, for an additional 24 h. After treatments, cells were washed with PBS, applied onto multi-spot microscope slides, and air-dried. Slides were then incubated with 2% paraformaldehyde (Electron Microscopy Science) for 30 min and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, Burlington, MA, USA) for 5 min. After three washes, cells were incubated with 1% glycine (Serva, Heidelberg, Germany), 3% BSA for a further 30 min. Thereafter, cells were incubated with mouse primary monoclonal antibody anti-γ-H2AX (phosphor-Ser 139) (sc-517; Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. Subsequently, cells were incubated with a polyclonal conjugated-Cy3 sheep anti-mouse antibody (Jackson ImmunoResearch, Cambridge, UK) for 30 min at room temperature and stained with DAPI (Sigma-Aldrich) for 1 min at room temperature. Slides were analyzed with Apotome Axio Observer Z1 inverted microscope (Zeis, Wetzlar, Germany) equipped with an AxioCam MRM Rev.3 at 40× magnification. Foci number analysis was performed by Image J software (1.47 version, NIH, Bethesda, MD, USA).