Reconstituting 60S Ribosome UFMylation

60S ribosomes were a generous gift from the Puglisi lab, and were also purified in‐house as described previously (Johnson et al, 2019 (link); Lapointe et al, 2021 (link)).For reconstituting 60S Ribosome UFMylation, approximately 50 nM purified 60S ribosomes were mixed with, 0.5 μM UBA5, 1 μM UFC1, 0.5 μM UFM1, and 100 nM UFL1/UFBP1 in a reaction buffer containing 25 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl₂ and 5 mM ATP and incubated at 30°C for 10 min or indicated time duration. The reaction was stopped by the addition of SDS loading buffer (1× final) and run on a 4–12% SDS–PAGE gel under reducing conditions followed by immunoblotting using indicated antibodies.
Ribosome UFMylation assay as described in Fig 4E was performed at 30°C with 1 μM UFC1, 0.5 μM UBA5, 0.5 μM UFM1, 0 nM or 75 nM UFL1/UFBP1, 50 nM purified 60S ribosomes, and increasing concentrations of CDK5RAP3 (0, 38, 75, 150 or 375 nM) in a reaction buffer of 25 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl₂ and 5 mM ATP. The reaction was quenched by the addition of SDS‐Load buffer (1× final) with reducing agent. Western blots show 10 min reaction time; immunoblots for RPL26 (Abcam, 59567) and UFL1 (Bethyl, A303‐455M) were run on the same gel, which was cut and probed for these proteins separately. Immunoblots for CDK5RAP3 (Bethyl, A300‐871A) and UFM1 (Abcam, Ab109307) were run on separate gels.