The slides Paclitaxel NSC 125973 were rinsed three times in PBS and treated with the SlowFade Antifade Kit according to the manu facturers specifications. Long lived protein degradation assay The long lived protein degradation assay was modified from published procedures. Cells were plated at 40,000 cells per well on 12 well plates. Long lived pro teins were labeled by removing the media, rinsing the cells once with PBS and culturing in the presence of 1 ml leucine free media containing 10% serumand 5 uCi ml leucine for 48 hours. After the labeling media was removed, unincorporated radioisotopes Inhibitors,Modulators,Libraries and degraded amino acids were removed by rinsing the plate three times with PBS. Short lived proteins were depleted by culturing the labeled cells with 1 ml Opti MEM con taining 4% serum and 2 mM cold leucine for 24 hours.

The chase medium was removed, cells were rinsed once with PBS and additives were added in Opti MEM 4% serum. Aliquots of the medium were removed at 24 hours, BSA was added to 3 mg ml final concentration and trichloroacetic acid was added to 10% final concentration. Proteins were precipitated by incubating at 4 C for 1 hour. Precipitates were recovered by centri fugation at 15,000g Inhibitors,Modulators,Libraries for 5 minutes at 4 C. Supernatants were collected and pellets were washed with cold 20% TCA. The washes were combined with the supernatants and this fraction represented small cleaved protein frag ments. PBS containing 0. 5% Triton X100 was added to the cells on the plate in order to recover counts asso ciated with the cells. After a 1 hour incubation, the lysate was removed and the wells were rinsed with PBS containing 0.

5% Triton X100. TCA precipitations Inhibitors,Modulators,Libraries were then performed on the protein lysates as described above. Finally, SDS PAGE lysis buffer was added to the wells to collect any remaining counts. TCA precipitates were air dried and then resuspended in 0. 1 N NaOH prior to counting. Aliquots of all fractions Inhibitors,Modulators,Libraries were counted with a scintillation counter. Proteolysis was determined as the ratio of non TCA precipitable counts to the total counts in each well. Cell based proteasome activity assay Cells were plated in black 96 well plates at 5,000 cells per well. They were cultured in Opti MEM containing 4% serum with chloroquine 1 hour prior to adding Inhibitors,Modulators,Libraries 10 ng ml TNFa. After 22 hours, the media were removed and cells were cultured in EBSS buffer containing the indicated additives.

For the 2 hour proteasome assays, there was no prior treatment with the additives in Opti MEM. Where indicated, epoxomicin an inhibitor of the chy motrypsin like activity of the 20S proteasome and calpain inhibitor X1 were added for 1 hour prior to the addition of TNFa. Proteolytic activity was determined by the addition Volasertib PLK inhibitor of the synthetic peptide Suc Leu Leu Val Tyr AMC prepared in EBSS and digitonin directly to the wells such that the final concentration of substrate was 50 uM and that of digitonin was 13. 3 ug ml.