Horseradish peroxidase antibody conjugates

Total cell lysates were prepared and analyzed by Western blot as described previously [20 (link)]. We used the following primary antibodies—for the detection of cleaved caspase 3: monoclonal antibody (MAB10753) from MilliporeSigma, St. Louis, MO, USA) or monoclonal antibody (SC-271028) from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and for PARP-1: SC-56196 from Santa Cruz (specific for the cleaved form) and #9542 from Cell Signaling Technology (Danvers, MA, USA) (recognizing full-length and cleaved PARP-1). Horseradish peroxidase-antibody conjugates (i.e., secondary antibodies) were obtained from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA). All antibodies were used according to the suppliers’ recommendations. For detection, SuperSignal West Pico PLUS Chemiluminescent Substrate was used (ThermoFisher Scientific, Waltham, MA, USA). Immunoblots were repeated to confirm the results. The uncropped Western blot images can be found at Figure S7.

Total cell lysates were analyzed by Western blot analysis as described earlier [41 (link)]. We used the following primary antibodies. For the detection of MGMT, we used a polyclonal antibody (#2739) from Cell Signaling Technology (Beverly, MA, USA) and a monoclonal antibody (ab39253) from Abcam (Cambridge, MA, USA). Anti-MSH2 (#2017) and anti-MSH6 (#5424) antibodies were from Cell Signaling. Human TRA-1-85 antibody (MAB3195) was from R&D Systems (Minneapolis, MN, USA) and beta-actin antibody (sc-47778) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase-antibody conjugates (i.e., secondary antibodies) were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). All antibodies were used according to the suppliers’ recommendations. All immunoblots were repeated at least once to confirm the results. Band intensities of MGMT were quantified using ImageJ, normalized relative to the respective actin control bands, and expressed in relation to the strongest reference band.

Cells were harvested by centrifugation, and cell pellets were lysed with RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitors. After the determination of protein concentration, we used the Western blot procedure to analyze the lysates according to previously published protocols [47 (link)]. The primary antibodies were as follows: In order to detect the MGMT protein, a polyclonal antibody from Cell Signaling Technology (Danvers, MA, USA; #2739) was used. For cleaved caspase 3, we used a monoclonal antibody (MAB10753) from Millipore-Sigma (Burlington, MA, USA). For EBNA-1, Ea-D, Zta/ZEBRA, CHOP, TRA-1-81, and beta-actin, we used monoclonal antibodies (sc-81581, sc-58121, sc-53904, sc-166682, sc-21706, and sc-47778), which were from Santa Cruz Biotechnology. As secondary antibodies, we applied horseradish peroxidase-antibody conjugates (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) according to the supplier’s recommendations. All immunoblots were repeated at least once to confirm the results. Uncropped images of Western blots along with molecular weight markers and the densitometric scanning of images are provided in the Supplementary Materials.