Rabbit anti mth1

The following antibodies were used in this study: mouse anti-β-actin (Abcam, Cambridge, UK; cat# ab6276), mouse anti-γH2AX-S139 (Millipore, Burlington, MA, USA; cat# 05-636), mouse anti-c-Myc (Santa Cruz, Dallas, TX, USA; cat# sc-42), rabbit anti-cleaved PARP Asp214 (Cell Signaling, Danvers, MA, USA; cat# 9541), rabbit anti-MTH1 (Novus Biologicals, Centennial, CO, USA; cat# NB100-109), rabbit anti-p53 pS15 (Cell Signaling; cat# 9284), mouse anti-p53 (Santa Cruz; cat# sc-126), mouse anti-GAPDH (Abcam; cat# ab8245), rat anti-RPA32 (Cell Signaling; cat# 2208).
The secondary antibodies used were: goat anti-rat Alexa Fluor^® 568 (Life Technologies, Carlsbad, CA, USA; cat# A-11077), goat anti-rat Alexa Fluor^® 647 (Life Technologies; cat# A-21247), IRDye^® 800CW donkey anti-rabbit (LI-COR, Lincoln, NE, USA; cat# 926-32213), IRDye^® 680RD donkey anti-rabbit (LI-COR; cat# 926-68073), IRDye^® 800CW donkey anti-mouse (LI-COR; cat# 926-32212), IRDye^® 680RD donkey anti-mouse (LI-COR; cat# 926-68072).

To analyse the effect of MTH1 on the sensitivity of cells to the alkylating agent Temozolomide, which exerts its toxicity primarily through methylation of O6-guanine, we made use of two glioblastoma cell lines, U251 and U251-MTH1 in which the MTH1 gene had been deleted using CRISPR/Cas9. These two cell lines were kind gifts from Dr Massimo Squatrito. MTH1 knockout was verified using western blotting using rabbit anti-MTH1 (Novus Biologicals). Cells were seeded in MEM medium (Gibco), 10% FBS (Gibco), 50 μg/ml Pen/Strep (Gibco), 1% MEM Non-Essential Amino Acids Solution (100×) (Gibco) and 1 mM Sodium pyruvate (Gibco) and treated with DMSO, Temozolomide (15 μM, Sigma-Aldrich) or Lomeguatrib (20 μM, selleckchem.com) or with both Temozolomide (15 μM) and Lomeguatrib (20 μM). Caspase 3/7 activity was monitored as a measure of early apoptosis after 48 h of treatment (800 cells were seeded in each well of a 384-well plate) at eight different Temozolomide and Lomeguatrib concentrations ranging from 0 to 100 μM, using the Caspase-Glo 3/7 assay (Promega) according to the manufacturer's recommendations. For assessment of cell viability, 400 cells were seeded in each well of a 384-well plate and Temozolomide and Lomeguatrib were added as described for the apoptosis assay. Cell viability was measured after 96 h using Resazurin as previously described (22 (link)).

Following antibodies were used: mouse anti beta-Actin (Abcam, ab6276, 1:1000), mouse anti-H2A.X phospho S139 (Millipore, 05-636, 1:1000), mouse anti-Histone H3 phospho-S10 (H3-pS10; Abcam, ab5176, 1:1000), rabbit anti-cleaved PARP (Cell Signaling, #9541, 1:1000), rabbit anti-PDL1 (Cell Signaling, #13684, 1:1000), rabbit anti-pTBK1 (Cell Signaling, #5483, 1:500), rabbit anti-TBK1 (Cell Signaling, #3504, 1:1000), rabbit anti-pSTING (Cell Signaling, #50907, 1:500), rabbit anti-STING (Cell Signaling, #13647, 1:1000), rabbit anti-cGAS (Cell Signaling, #15102, 1:1000), rabbit anti-MTH1 (Novus Biologicals, NB100-109, 1:1000). Secondary antibodies were: Peroxidase AffiniPure Donkey Anti-Rabbit IgG (Jackson ImmunoResearch, 711-035-152, 1:5000), Peroxidase AffiniPure Donkey Anti-Mouse IgG (Jackson ImmunoResearch, 715-035-150, 1:5000), IRDye 680RD Goat Anti-Mouse IgG (LI-COR, 926-68072, 1:5000) and IRDye 800CW Donkey Anti-Rabbit IgG (LI-COR, 926-32213, 1:5000).