Experimental Conditions

Four biological replicate experiments were performed. HCT116 cells metabolically labeled with heavy lysine were and treated with 1uM bortezomib (Btz) Equal amount of untreated cells were mixed. Ratios are oriented heavy to light. Reference: Figure 1

HCT116 cells labeled with heavy lysine were treated with MLN4942 for 0, 2, 4, or 8 hours and mixed with equal amount of untreated cells. Ratios are oriented heavy to light. Reference: Figure 2, 6

Unlabeled HCT116 cells were grown in media containing heavy lysine for 4 hours (pulse) followed by either continued unperturbed growth in heavy lysine media (chase) or in the presence of Btz with and without the translational inhibitor cyclohexamide (CHX)

Cells treated with Btz for 8 hours were either metabolically labeled and treated with Usp2cc, the catalytic domain of ubiquitin specific protease 2, or mock treated. Ratios are oriented heavy to light.

HCT116 cells were treated with Btz for 8 hours in the presence or absence of CHX.

Metabolically labeled cells were treated for 8 hours with either Btz or epoxomicin, an inhibitor with distinct preference for proteasome active sites.

Heavy labeled cells were treated with Btz for increasing amounts of time. At each time point, heavy cells were mixed with light untreated cells.