However, there are many problems with this interpretation. First, bortezomib did not inhibit purified PSAP, which was the major Ala-AMC and Leu-AMC-cleaving aminopeptidase detected in HEK293T cell extracts. Second, LEE011 CDK inhibitor although MG262 and MLN2238 also inhibited the HEK293T cell aminopeptidase activity and purified PSAP, only MG262 caused a large increase in many of the intracellular peptides; MLN2238 did not show this effect. Finally, neither bestatin nor bestatin methyl ester caused a large change in the levels of intracellular peptides. The absence of large changes in peptide levels in response to treatment with these inhibitors suggests that neither PSAP nor other bestatin-sensitive enzymes contribute to the degradation of the intracellular peptides observed in this study. This finding is consistent with the observation that mice lacking either LAP or PSAP show normal processing and presentation of peptides in complex with MHC class I molecules. Previous studies investigating peptides bound to MHC class I molecules tested the origin of these peptides by treating cells with TH-302 proteasome inhibitors and measuring levels of HLA-bound peptides. One study found 104 different peptides bound to HLA-B27, and although the majority was decreased by treatment of cells with epoxomicin, 31 peptides were not affected more than 20% and were therefore considered to be proteasome independent. A subsequent study examining peptides bound to other HLA proteins also found a substantial number of peptides that were not affected by treatment with either epoxomicin or MG132. Many of these proteasome-independent peptides arose from small basic proteins. In the present study, only three peptides were consistently found to be resistant to the various proteasome inhibitors. The three proteins that give rise to the peptides in Table 2 range in size from 63 to 272 amino acids. This is comparable to the size range of the proteins listed in Table 1 and Table 3. Furthermore, basic proteins are not more common than 
acidic proteins in Tables 2 and 3. Therefore, the tendency for proteasome-independent HLA-bound peptides to be products of basic proteins is not shared by the proteasomeindependent peptides found in whole cell extracts in the present study. On the other hand, all of the proteins listed in Tables 1�C3 are under 300 amino acids in length, which is well below the size of the average protein encoded by the human genome. Milner and colleagues examined the effect of epoxomicin and bortezomib on the rate of synthesis of HLA-bound peptides and cellular proteins in MCF-7 cells. Although the rate of synthesis of many HLA-bound peptides was decreased when cells were treated with the proteasome inhibitors for 4 hours, other peptides showed no effect or even an increase in their rates of synthesis in response to the proteasome inhibitors. Similarly, the rate of cellular protein synthesis was generally decreased for most proteins, but some were not affected or had elevated rates of synthesis. A comparison of the proteins listed in the supplemental data Table S2A of Milner et al with the proteins found in the present study revealed ten proteins in common for which data were available for both epoxomicin and bortezomib. Two of these proteins showed a decrease in levels of intracellular peptides in our analysis and also a decrease in protein synthesis. Another protein showed a decrease in intracellular peptides and protein synthesis with epoxomicin and no substantial change with bortezomib. However, none of the other seven proteins showed a correlation between the rate of protein synthesis and the levels of intracellular peptides after treatment with bortezomib or epoxomicin; gene names of these proteins are PPIA, TMSB10, EIF5A, ERH, MIF, UBA52, and RPLP2.