Mg132 bml pi102 0005

Endogenous Fbw7α was immunoprecipitated at 4°C for 20 min from MII-arrested egg extracts (50 μl) or for 90 min from 500 μg of proteins from HeLa cell lysates using polyclonal anti-Fbw7 antibodies and protein A magnetic Dynabeads (Thermo Fisher Scientific). After several washes with XB buffer, bead pellets were boiled in SDS sample buffer, separated by 7% SDS-PAGE and transferred onto Immobilon-P membranes (Millipore). Endogenous Fbw7α was visualized with anti-Fbw7 or anti-pS18 antibodies and HRP-conjugated protein A (Thermo Fisher Scientific) was used as a second step, except in Fig 1B, where TrueBlot^® anti-rabbit Ig IP beads (eBioscience) were used. For co-immunoprecipitation of ectopically expressed proteins, HeLa cells were co-transfected with the corresponding constructs. Cells were homogenized in lysis buffer: 50 mM K-HEPES pH 7.7, 100 mM KCl, 1 mM EDTA, 1% Triton X-100, 1 mM DTT, 10% glycerol, 1× protease inhibitor and phosphatase inhibitor cocktail (Roche Diagnostics) and 25 μM MG132 (BML-PI102-0005, Enzo Life Sciences) at 4°C for 30 min. After centrifugation at 12,000 g for 20 min, 2 μg anti-HA or anti-FLAG antibodies were added to the pre-cleared supernatants. Ectopic Fbw7α proteins were immunoprecipitated at 4°C for 1 hr from 500 μg to 1 mg of protein cell lysates and collected on protein-A or -G magnetic beads.

DUBs-IN-2 (HY-50737A), ML323 (HY-17543), ML364 (HY-100900), P22077 (HY-13865), Spautin-1 (HY-12990), IU1 (HY-13817), LDN-57444 (HY-18637), PR-619 (HY-13814), BAY 11-7082 (HY-13453), MF-094 (HY-112438), IKK 16 (HY-13687) were purchased from MedChemExpress. TCID (S7140) was purchased from Selleck. MG132 (BML-PI102-0005) was purchased from Enzo life science. Cycloheximide (C7698-5G) was purchased from Sigma. Anti-mouse PD-1 (clone 29F.1A12)/PD-L1 (clone 10 F.9G2) for mice treatment antibodies were provided by the Laboratory of Dr. Gordon J. Freeman.