Anti ph2ax ser139

Immunoblot analysis was performed as described previously (16 (link)). For studying the PARP trapping effect of the PARPi, isolation of the nuclear soluble and chromatin bound fraction was performed using the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Fisher Scientific 78840) according to the manufacturer's protocol. Cells were therefore treated with 0.005% Methyl methane sulfonate (MMS, Thermo Fisher Scientific, 156890050) and 2 μmol/L talazoparib. The following primary antibodies were used for immunoblot analysis: anti-p21 Waf1/Cip1 (DCS60; Cell Signaling Technology 2946, 1:1,000), anti-pH2AX (Ser139; Merck, JBW301, 1:500), anti-β-Actin (Cell Signaling Technology 4967, 1:2,000), anti-p53 (DOI; sc-126, 1:500), anti-ATM (D2E2; Cell Signaling Technology 2873, 1:1,000), anti-PARP (Cell Signaling Technology 9542, 1:1,000), and anti-H2A.X (Cell Signaling Technology 2595, 1:1,000). The following secondary antibodies were used: anti-mouse IgG, horseradish peroxidase (HRP)-linked (Cell Signaling Technology 7076) and anti-rabbit IgG, HRP-linked (Cell Signaling Technology 7074).

Processing, embedding and sectioning of formalin-fixed ID8-NGL tumor tissue, and hematoxylin and eosin staining for histology, were performed in The Allergy/Pulmonary & Critical Care Med Division Immunohistochemistry Core at Vanderbilt [27 (link)]. Immunofluorescence analysis of formalin-fixed paraffin-embedded tumor tissue or methanol-fixed cultured ID8-NGL cells was performed using standard techniques [21 (link), 19 (link)]. The following primary antibodies: mouse monoclonal anti-pH2AX(ser139) (EMD Millipore, Cat# 05-636, 1:250 dilution); rabbit polyclonal anti-Ki67/Mib-1 (Abcam, Cat# ab16667; 1:200 dilution); and rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling Technology, Cat# 9661; 1:100 dilution), were used. Secondary antibody used was goat anti-rabbit Alexa Fluor 488 (Life Technologies, Cat# 11070) (all 1:200 dilution). Images were acquired and analyzed as previously described [21 (link), 19 (link)]. For quantifying the percentage of, where applicable, tumor cells or macrophages positive for these proteins, at least 5 independent fields were assessed with at least 200 cells counted per sample.