Mln2238

High glucose Dulbecco’s Modified Eagle’s Medium (DMEM), L-glutamine enriched Roswell Park Memorial Institute medium (RPMI) and Dulbecco’s Phosphate Buffered Saline (DPBS) were purchased from Invitrogen. Hydroxylamine, glycine, sodium hydroxide, dibasic sodium phosphate and dimethyl sulfoxide (DMSO) were obtained from Sigma. Acetonitrile was obtained from Fisher. Hydrochloric acid, trifluoroacetic acid (TFA) mass spectroscopy grade and C-18 spin columns were purchased from Pierce Thermo Scientific. MG132, MG262 and clasto-Lactacystin β-lactone were purchased from Boston Biochem. AM114, butabindide and puromycin were purchased from Tocris Bioscience. Other inhibitors and their commercial sources were bortezomib (LC Laboratories), MLN2238 (Selleckchem), carfilzomib (ChemieTek), bestatin (Sigma), and bestatin methylester (Calbiochem). Recombinant human puromycin-sensitive aminopeptidase was purchased from R&D Systems. Suc-Leu-Leu-Val-Tyr-AMC, Ala-Ala-Phe-AMC, Leu-AMC and Ala-AMC were procured from Bachem. The isotopic labeling reagents 4-trimethylammoniumbutyryl-N-hydroxysuccinimide (TMAB-NHS) containing either 0, 3, 6, or 9 atoms of deuterium (D0-, D3-, D6-, and D9-TMAB-NHS, respectively) or 9 atoms of deuterium and three ¹³C atoms (D12-TMAB-NHS) were synthesized as described [30] .

Bortezomib, MLN2238, nocodazole and RO-3306 were purchased from SelleckChem (Houston, TX) for the in vitro studies. Compounds were resuspended in DMSO and frozen down in 20 microliter aliquots to limit freeze-thaw cycles. Compounds were added as noted in the figure legends. In vitro studies used 15 nM for Bortezomib and 100 nM for MLN2238 or are otherwise specified in the text. For the pulse experiments, we used 2.5 micromolar of MLN2238.

All antibodies were purchased from Cell Signaling. Bortezomib, LBH589 (panobinostat), and MLN2238 (ixazomib) were purchased from Selleck Chemicals and suspended in DMSO.