To begin the western blotting of the normalized digested DNA samples, add 4X Laemmli sample buffer and boil the sample for five minutes. Load five to six micrograms of digested sample onto 4 to 20%polyacrylamide gel and perform SDS-PAGE to resolve unmodified and post-translational modifications or PMT conjugated DPCs. Incubate the gel with Coomassie blue stain overnight at room temperature.
Wash the gel with double distilled water for two hours before image acquisition. To detect ubiquitylation, SUMO-1 or SUMO-2 and 3 modification, ADP ribosylation, total TOP1, TOP2 alpha, or TOP2 beta DPCs, dilute the corresponding antibodies as shown in the table. Next, transfer the gel in an appropriate dilution of primary antibody in blocking buffer and incubate the membranes overnight at four degrees Celsius.
Once done, incubate PBST washed membrane with a secondary antibody diluted 5, 000 fold in blocking buffer for 60 minutes at room temperature before developing the membrane with enhanced chemiluminescence reagent. Acquire an image using the imaging system. The camptothecin exposure induced TOP1 DPCs formation.
The TOP1 DPCs formation peaked after 20 minutes of camptothecin exposure similar to the anti-SUMO-2 and 3 modification. The culmination of SUMO-1 modification and ubiquitylation was also observed. DPCs and their SUMO-2 and 3 TOP1 modifications diminished in a camptothecin dose dependent manner.
Adipocyte-induced TOP2 DPCs and SUMO-2 and 3 modification reached to peak at 20 minutes and then decreased. In comparison, SUMO-1 and ubiquitin modifications peaked at 60 minutes. The formaldehyde-induced DPCs and their SUMO-2 and 3, SUMO-1, and ubiquitylation formed and accumulated in a dose dependent manner.
The quantitative detection of PARylation of TOP1 DPCs using an anti-PAR antibody, TOP1 DPC PARylation was not detectable unless a PARG inhibitor was added to the cell, suggesting that PARylation transpires promptly and is highly dynamic.
