Lactacystin

U373-MG cells were starved of methionine and radiolabeled for 5–10 min with 100 μCi/ml of [³⁵S] Met. Following radiolabeling, cells were either chased with 2 mM methionine-supplemented media or immediately prepared for lysis as follows. Cells were washed twice with chilled PBS and lysed in RIPA buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% SDS, 10 mM iodoacetamide, and 1x proteasome inhibitor cocktail (P8340, Sigma Aldrich)). The lysates were rocked for 30 min then centrifuged for 10 min at 11,000 rpm to remove nuclei and insoluble material. Supernatant fractions were treated with 10 μg w6/32, 10 μg HC10, and 10 μg HCA2 to isolate MHC class I and with 20 μl anti-calnexin antiserum as a radiolabeling and immunoisolation recovery control. After 2 h a mix of protein A-Sepharose (GE Healthcare) and protein G-Sepharose (Life Technologies) beads was added and incubated for 1 h. Beads were washed four times with 0.5% Nonidet P-40, 10 mM Hepes pH 7.4, 150 mM NaCl and then proteins were eluted, separated by SDS-PAGE under reducing conditions and visualized by fluorography. For inhibition of proteasomal activity, cells were pre-treated for 60 min with 25 μM lactacystin (Abcam), starved of methionine in the presence of 25 μM MG132 (Boston Biochem), radiolabeled in the presence of both inhibitors, and chased in the presence of 25 μM MG132.

Confluent MLO‐A5 cell cultures were treated with media supplemented with 0–10 μM N‐(benzyloxycarbonyl)‐Leu‐Leu‐Leucinal (MG132), lactacystin (Cayman Chemicals), Bortezomib (Velcade®) (Santa Cruz), and Withaferin‐A (Abcam, Cambridge UK) and then incubated at 37°C for 24 h. Confluent primary cell cultures were treated with media supplemented with ALLN, MG132, and lactacystin. Thereafter, protein/RNA was extracted from monolayers, as described below, for the determination of E11 protein/mRNA expression.