Proteasome inhibitor cocktail

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.

Prostate cancer cells were lysed by RIPA buffer containing proteasome inhibitor cocktail (Sigma) or performed nucleocytoplasmic fractionation according to the manufacturer’s instructions (G-Biosciences). The samples were analyzed by immunoblotting with primary antibodies to: PARP (Cell Signaling Technology Cat# 9532, 1:1000), cleaved-caspase 3 (Cell Signaling Technology, Cat# 9664, 1:1000), γH2A.X (Cell Signaling Technology Cat# 2577, 1:1000), AR (Santa Cruz Biotechnology Cat# sc-7305, 1:1000), PSA (Cell Signaling Technology Cat# 5365, 1:1000), UBE2C (Cell Signaling Technology Cat# 14234, 1:200), E2F1 (Cell Signaling Technology Cat# 3742, 1:1000), FOXA1 (Cell Signaling Technology Cat# 53528, 1:1000), Ku70 (Cell Signaling Technology Cat# 4588, 1:1000), Ku80 (Cell Signaling Technology Cat# 2180, 1:1000), BRCA1 (Cell Signaling Technology Cat# 9009, 1:1000), Rad51 (Cell Signaling Technology Cat# 8875, 1:1000), Lamin B (Cell Signaling Technology Cat# 13435, 1:1000), and GAPDH (Santa Cruz Biotechnology Cat# sc-47724, 1:1000).

Protein expression was assessed by Western blotting. Cells were harvested from the culture plates and total cellular proteins were extracted by lysis buffer containing 1% Triton X-100, 1% proteasome inhibitor cocktail (Sigma-Aldrich). Lysates were cleared by centrifugation at 13,000 g for 20 min and protein concentrations were measured using the BC Assay Protein Quantitation Kit (Uptima, Oakland, CA). Total protein lysates (50 μg) were fractionated on 15% SDS-PAGE then transferred to nitrocellulose membranes (GE Healthcare Life Sciences, Vélizy-Villacoublay, France). Membranes were blocked for 1 h in PBST with 2.5% BSA, and then incubated with primary antibodies overnight at 4 °C, rabbit polyclonal anti-BDNF (sc-546, Santa Cruz [51 (link)]) at 1:200 and mouse monoclonal anti-α-tubulin (Clone DM1A, Sigma-Aldrich) at 1:10,000. Incubation with secondary antibodies conjugated to infrared fluorophores (goat anti-rabbit IgG Dylight 800 and anti-mouse IgG Dylight 680 at 1:10,000 from Thermo Scientific) was performed for 1 h. An Odyssey infrared imaging system (LI-COR, Bad Homburg, Germany) was used to scan membranes at a wavelength of 680 nm (anti-mouse) or 800 nm (anti-rabbit). Data were analyzed with Image Studio 1.1 software (Li-COR).