Mln4924

The stable U2OS cell lines expressing myc-BirA∗-DCAF, myc-BirA∗-DDB1, and GFP-DDB1 were generated using the Flp-In T-Rex system (Thermo Fisher Scientific) using respectively pGLAP1-myc-BirA∗-DCAF, pGLAP1-myc-BirA∗-DDB1, and pGLAP1-GFP-DDB1 constructions. U2OS Flp-In-Rex (U2OS-FT) cells were maintained at 37 °C, 5% CO₂ in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific), supplemented with 10% FB essence (Wisent, St John’s), 50 U/ml penicillin/streptomycin, and 10 mM Hepes. U2OS-FT cells were transfected using Lipofectamine LTX (Thermo Fisher Scientific) during 48 h in 6 cm Petri dishes with 4.5 μg of Flp-Recombinase expression vector pOG44 (Thermo Fisher Scientific) and 500 ng of plasmid DNA. Transfected cells were selected for 2 weeks with hygromycin (50 μg/ml, Thermo Fisher Scientific) and blasticidin (10 μg/ml, Wisent). The expression of the cDNA was achieved by adding 10 μg/ml doxycycline (Clontech Laboratories, Mountain View) in the medium for 24 h or 48 h (or not as control). Cells were lysed directly in Laemmli sample buffer, and the extracted proteins were resolved by SDS-PAGE prior to being transferred to a nitrocellulose membrane. Immunoblotting was performed with a BirA∗ antibody (Novus Biologicals #6C4c7, 1:1000 dilution) or GFP (Santa Cruz Sc-9996, 1:1000 dilution). To inhibit cullins, we treated cells 24 h with 10 μM MLN4924 (Cell signaling 85923S).