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. 2017 Mar 20;114(14):3565–3571. doi: 10.1073/pnas.1700949114

Fig. S5.

Fig. S5.

(Related to Fig. 6) (A) Lenalidomide-induced degradation of CRBN neosubstrates requires p97. U266 cells were pretreated with the indicated doses of CB-5083 or NMS-873 for 30 min, followed by the addition (or not) of lenalidomide (Len) (10 μM) for 4 h. Cell lysates were fractionated by SDS/PAGE and immunoblotted for the indicated endogenous proteins. (B) Depletion of p97 or proteasome activity promotes the accumulation of ubiquitylated IKZF1, suggesting that p97 functions downstream of IKZF1 ubiquitylation. HEK293 cells were transfected with plasmids encoding HAUb and IKZF1Flag. After 24 h of transfection, the cells were pretreated with 20 µM MG132 or 10 µM CB-5083 for 30 min followed by the addition (or not) of 10 µM lenalidomide for 3 h. Denatured lysate proteins were immunoprecipitated with anti-Flag antibody. The input lysates and bound fractions were evaluated by SDS/PAGE and immunoblotting with antibodies against the HA and Flag tags.