Western Blot Analysis of MGMT and Mismatch Repair Proteins

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.