Fig 1: PROTAC compounds degrade FER in a cereblon- and proteasome-dependent manner.A, OVCAR-5 cells or CAOV4 cells were treated with 10 nM 008, respectively, and harvested at the indicated time points for immunoblotting analysis. Total FER and actin were probed. B, results from (A) were represented as means ± SD from three independent replicates. The time–response nonlinear regression was drawn by GraphPad Prism. C, OVCAR-5 cells, in the presence of 10 µg/ml CHX, were treated with DMSO or 100 nM 008, followed by immunoblotting analysis. Total FER and actin were probed. D, OVCAR-5 cells were treated with DMSO or compound 008 for 16 h and then subjected to mass spectrometry analysis. Each experiment was repeated three times. E and F, OVCAR-5 and CAOV4 cells were treated with 008 or 039, respectively, for 16 h. The protein levels of GAK and AAK1 were detected by the corresponding antibodies, as indicated. Expressions of negative control CHK2 and IRAK1 were also probed, with actin as a loading control. G, Bel7404WT and Bel7404CRBN-/- knockout cell lines were treated with 008 and 039 for 16 h. The protein levels of FER, GAK, AAK1, CHK2, IRAK1, and loading control actin were checked with immunoblotting analysis. H–K, OVCAR-5 cells were treated with the proteasome inhibitor MG132 or the lysosome inhibitor CQ in combination with compounds 008 (H) or 039 (I) for 4 h, as indicated. Cell lysates were harvested and immunoblotted with an FER antibody, using actin as a loading control. Results represented means ± SD from three replicates (J and K). ***p < 0.001 and ****p < 0.0001. AAK1, AP2-associated kinase 1; CHX, cycloheximide; DMSO, dimethyl sulfoxide; FER, Fps/Fes Related; GAK, cyclin G-associated kinase; PROTAC, PROteolysis TArgeting Chimera.