Fig 2: CRBNs is a transmembrane protein-specific co-chaperone of the HSP90-AHA1 axis(A) Immunoblot analysis of TM proteins identified in the cell surface analysis (left panel) and known non-TM HSP90 clients (right panel) from MM1S cells that were treated with 10 µM lenalidomide or 1 µM 17-AAG for 72 h before harvest.(B) Representative immunofluorescence images of HeLa cells that were transfected with constructs encoding HA-CD98hc and FLAG-LAT1. The cells were stained with antibodies to HA (green) and FLAG (red). DNA was stained with DAPI (blue). Cells were treated with 50 µM lenalidomide, 50nM 17-AAG, and DMSO for 16 h as indicated. Scale bars, 10 µm. Quantification is presented in Figures S6D and S6E.(C) IP analysis of FLAG-CD98hc from HEK293T cells that were treated with cycloheximide (CHX) for the indicated times before harvest to block de novo protein synthesis.(D) IP analysis of FLAG-LAT1 from HEK293T cells that were processed and analyzed as in (C).(E) Immunoblot analysis of MM1S cells, in which AHA1 was lentivirally silenced using the indicated shRNAs.(F) Schematic representation of CRBNs function toward TP clients in the absence and presence of IMiDs.

Fig 3: CRBN interacts with the ATP-bound conformation of HSP90(A) Endogenous IP of CRBN from U266 cells and analysis of co-purified endogenous proteins as indicated. The asterisk marks an unspecific band. LE, long exposure; SE, short exposure.(B) IP analysis of different FLAG-HSP90? mutants transfected into HEK293T cells (right). Models of HSP90 dimer conformations induced by the indicated mutations (left).(C) IP analysis of FLAG-HSP90? transfected into HEK293T cells. Lysis and IP was performed in the absence or presence of 20 mM sodium molybdate.(D) Sequential FLAG/HA IP from HEK293T WCE transfected with the indicated HSP90? constructs. After lysis and FLAG IP, bait was eluted with FLAG peptide and used for an HA IP, resulting in complexes consisting of one FLAG- and one HA-tagged HSP90 protomer. The second lane contains complexes with both unmutated promoters; in the third lane, one protomer carries a V410E mutation.(E) IP analysis of FLAG-HSP90? transfected into CRBN-/- HEK293T WCE reconstituted with CRBNWT or CRBNYW/AA as indicated. Cells were pretreated with lenalidomide (50 µM), 17-AAG (1 µM), or DMSO for 24 h prior to harvest.(F) Quantification of IP analysis presented in (E) (n = 3 independent experiments).Data are mean ± SD. **p < 0.01 and ***p < 0.001 by one-sample t test.

Fig 4: CRBN modulates HSP90-AHA1 activity toward client proteins(A) IP analysis of FLAG-CRBN from HEK293T cells with co-expression of either AHA1WT or AHA1Y223E using low-salt buffer.(B) IP analysis of FLAG-CRBNWT or CRBNYW/AA from CRBN-/- HEK293T WCE in low-salt buffer. Cells were treated with DMSO or lenalidomide (50 µM) for 24 h prior to harvest and immunoblot analysis.(C) IP analysis of FLAG-AHA1 mutants transfected into HEK293T cells using low-salt buffer.(D) IP analysis of FLAG-CRBN transfected into HEK293T cells, in which AHA1 was lentivirally silenced using the indicated shRNAs.(E) Immunoblot analysis of HEK293T cells transfected with CFTR, FLAG-AHA1Y223E, and different CRBN constructs as indicated. Cells were treated with DMSO or lenalidomide (50 µM) for 24 h prior to harvest and immunoblot analysis.(F) Quantification of the immunoblot analysis (lanes 2–4) presented in (C) (n = 3 independent experiments).(G) In vitro ATPase activity of HSP90 with addition of CRBN, AHA1WT purified from 293T cells. Lenalidomide (1 µM) was added as indicated. Purified proteins are shown in Figure S4H.Data in this figure are mean ± SD. **p < 0.01, ***p < 0.001, and ****p < 0.0001 by one-sample t test or Student’s t test.