To validate the mechanism of PROTAC mediated degradation of BTK, Mino cells were pre-treated for 2 hours with either Ibrutinib or thalidomide-OH, and subsequently treated with the PROTACs for an additional 24 hours. Both Ibrutinib pre-treatment as well as thalidomide-OH, hindered BTK degradation (Fig. ). In contrast to the covalent PROTACs, degradation by the non-covalent NC-1 was only slightly hindered by thalidomide. In addition, RC-1m, a methylated thalidomide analog of RC-1, no longer able to bind CRBN, lost all activity (Supp. Fig. ), further suggesting CRBN mediated degradation. We treated Mino cells with Bortezomib, a proteasome inhibitor , for 1 hour before treatment with the PROTACs and assessed BTK levels after an additional 4 hours. Bortezomib significantly inhibited degradation, suggesting proteasome-dependent degradation (Fig. ). A. Mino cells were either pre-treated for 2 hours with Ibrutinib/thalidomide-OH or untreated, before treatment with a BTK PROTAC for 24 hours. Subsequently BTK levels were measured via western blot.