Nimbolide

Covalent ligands screened against RNF114 were purchased from Enamine LLC, including EN219. Structures of compounds screened can be found in Table S1. See Supplementary Information for synthetic methods and characterization for EN219 and degraders. Nimbolide was purchased from Cayman Chemicals (Item No. 19230).

Recombinant Flag-tagged RNF114 was used as bait to precipitate pure recombinant p21 (Origene Technologies Inc., TP309752 and TP720567) using Anti-Flag agarose beads (GenScript Biotech Corp., L00432). One microgram of Flag-RNF114 was added to 50 μL of TBS, followed by the addition of nimbolide (100 μM final concentration, Cayman Chemical Co., 19230) or equivalent volume of DMSO. Samples were incubated at room temperature for 30 min. One microgram of pure p21 was added to each sample, and samples were incubated at room temperature 30 min with agitation. Ten microliters of Flag agarose beads were added to each sample, and samples were agitated at room temperature for 30 min. Washes (3 times, 1 mL TBS) were performed before proteins were eluted using 50 μL of TBS supplemented with 250 ng/μL 3 × FLAG peptide (ApexBio A6001). Supernatant (30 μL) were collected and after the addition of Laemmli’s reducing agent (10 μL), samples were boiled at 95 °C for 5 min and allowed to cool. Samples were analyzed by Western blotting as described above.