Fig. 6.

p97 is required for immunomodulatory drug-induced degradation of CRBN neosubstrates. (A) MM1S cells were pretreated with the indicated doses of CB-5083 or 1 µM MLN4924 (MLN) for 30 min, followed by the addition of lenalidomide (Len) (10 μM) for 4 h. Cell lysates were fractionated by SDS/PAGE and immunoblotted for the indicated endogenous proteins. The relative ratios of IKZF1:GAPDH or IKZF3, normalized to lane 1, are shown here and in B–D. (B) L363 cells were pretreated with CB-5083 (2 μM) for 30 min, followed by the addition (or not) of lenalidomide (10 μM) for 4 h. Cell lysates were fractionated by SDS/PAGE and immunoblotted for the indicated endogenous proteins. (C) MM1S cells stably expressing control (CT) or CRBN shRNAs were pretreated with CB-5083 (2 μM) for 30 min, followed by the addition (or not) of lenalidomide (10 μM) for 5 h. Cell lysates were analyzed by immunoblotting with the indicated antibodies. (D) As in C, except that cells were pretreated (or not) with NMS-873 (2 or 5 μM) for 30 min. (E) U937 and MM1S cells were pretreated (or not) with CB-5083 (2 or 10 μM), MLN4924 (1 μM), or MG132 (10 μM) for 1 h, followed by treatment with 10 nM CC-885 for an additional 2 h. Whole-cell extracts were subjected to immunoblot analysis. (F) Cells were pretreated with CB-5083 (2 μM) and/or lenalidomide (10 μM) for 30 min followed by the addition of cyclohexamide (CHX) (100 μg/mL). At the indicated times after the addition of cyclohexamide, samples were harvested for immunoblot analysis. The relative ratios of CK1α:GAPDH, normalized to lane 1, are shown.