Complete mini edta protease inhibitor cocktail

Hypoxanthine, sorbitol, percoll, MG132, Epoxomicin, Lactacystin, Clasto-Lactacystin β-lactone, Gliotoxin, MG115, EDTA, sodium phosphate dibasic, monosodium phosphate, Tris HCl, sodium chloride, Tween-20, sodium pyrophosphate, glycerol-2-phosphate, saponin, bovine serum albumin (BSA), dithiothreitol (DTT) and phenylmethylsulphonyl fluoride (PMSF) were purchased from Sigma. Glutathione acceptor beads, protein A donor beads, DELFIA enhancement solution and DELFIA secondary antibody (Eu-N1 rabbit Anti-mouse-IgG) came from Perkin Elmer. NP-40 was purchased from Calbiochem, sodium fluoride from Panreac, antibody anti-ubiquitin P4D1 from Santacruz, deubiquitylases inhibitor PR-619 came from Merck, complete mini EDTA protease inhibitor cocktail from Roche, antibody anti-ubiquitin FK2 from Enzo, biotin-TUBEs from Life sensor, RPMI 1640 medium from Gibco, AlbuMAX II from Invitrogen, bortezomib from Selleckchem, enhanced chemiluminescence (ECL) from GE Healthcare and PBS from Oxoid. Atovaquone was prepared in house.

P19 cells were cultured in 24-well plate in DMEM + 10% FBS, washed with methionine/cysteine-free DMEM, and supplied with freshly prepared medium containing [³H] methionine at a final concentration of 0.23 mCi/μmol in methionine/cysteine-free DMEM for 2 h. At the end of incubation, cells were washed twice in PBS containing 1 mg/ml of unlabeled methionine and lysed in 0.4 ml of RIPA buffer (20 mM sodium phosphate, 150 mM NaCl, 5 mM sodium pyrophosphate, 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 0.1% SDS, pH 7.4) supplemented with 1 tablet/10 ml of complete mini-EDTA protease inhibitor cocktail (Roche) and 1% BSA on ice for 20 min. The lysate was centrifuged at 12,000 × g for 15 min, an aliquot of 10 μl of the supernatant was taken for measurement of protein concentration by Bradford assay, the remaining lysate was precipitated by TCA at a final concentration of 10% at 4 °C for 10 min. Protein pellet was collected by centrifugation for 30 min at 12,000 × g at 4 °C, resuspended in 50 μl of 0.2 M NaOH, and measured for radioactivity by liquid scintillation counting.

Cells were lysed in cell lysis buffer (150 mM NaCl, 10 mM CaCl₂, 50 mM HEPES (pH 7.3), 0.5% Triton X-100, 0.5% NP-40, Complete Mini-EDTA Protease Inhibitor Cocktail (Roche)), and protein concentrations determined by Bradford assay. Samples containing equal protein were heated at 95°C for 5 minutes in Laemmli sample buffer containing 10% beta-mercaptoethanol and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), using 4–12% or 10% NuPage Bis-Tris pre-cast gels (Life Technologies), followed by immunoblotting using PVDF membranes (Life Technologies). Membranes were blocked for 30 minutes in 5% (w/v) non-fat milk and then sequentially incubated with primary and HRP-conjugated secondary antibodies. HRP activity was detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).