FIGURE 2.

Experimental strategy for analyzing DNA damage-associated ubiquitylation. Cells are metabolically labeled using heavy isotope containing lysine and arginine (SILAC) and subsequently treated with a DNA damage-inducing agent. Pretreatment with an inhibitor or knockdown of ubiquitin-modifying enzyme can be performed before treatment with the DNA damage-inducing agent. Proteins are extracted from cells and ubiquitylation is quantitatively investigated using ubiquitin remnant profiling and/or ubiquitin chain topology profiling.