For confirmation of direct target engagement, FLAG-tagged BRD2 and HA-tagged DCAF11 were expressed in Hep G2 cells. Co-immunoprecipitation of FLAG-BRD2 enriched DCAF11 in the presence of bifunctional compound 9, indicating that 9 mediates complex formation between BRD2 and DCAF11 (Fig. ). DCAF11 may expose three reactive cysteines in its putative small-molecule binding site , and  unsaturated indolinones embody a potential electrophilic Michael acceptor. Although, to the best of our knowledge, covalent targeting by GW5074 or sunitinib has not been reported, degrader 9 might covalently bind to one or several of the cysteines in DCAF11. Indeed, while ,-unsaturated degraders 9 and 10 displayed BRD2degrading activity, the corresponding saturated analogues 15 and 16 did not induce degradation of BRD2 (Fig. and Supplementary Fig. ). ) at 30 μM or MLN4924 at 1 μM for 1 h, followed by treatment with 9 (10 μM, 4 h) or DMSO as a control. Data are mean values ± SD (n > 3). h, Structures of representative indolinones (45, 46) and corresponding bifunctional compounds (47, 48). i, BRD2 levels in Jurkat cells treated with compound 9, 47 or 48 at 3 μM for 6 h. Data are mean values ± SD (n > 3). Statistical significance was calculated with unpaired two-tailed Student's t-tests comparing 10 to 47 or 48 (***P < 0.001). j, Influence on cell viability. Jurkat cells were treated with compounds for 3 days, and cell viability was determined by means of the CellTiter-Glo reagent. Data are mean values ± SD (n = 3). The data of GI50 are shown in Supplementary Fig. . k, Caspase-3/7 activity for apoptosis detection. Jurkat cells were treated with compounds as indicated for 18 h, and caspase-3/7 activity was measured using Caspase-Glo ® 3/7 Assay System. Data are mean values ± SD (n = 3). Full images of blots and gels are shown in Supplementary Fig. .