Validation of the antiproliferative activity of UBX-382 in diverse cell lines derived from hematological malignancies. (A) Inhibitory effects of ibrutinib (yellow circle), ARQ-531 (purple diamond), Thalidomide (hunter green triangle), Lenalidomide (sky blue inverted triangle), and UBX-382 (red square) on cell proliferation in the examined cell lines at the indicated increasing doses were monitored for 5 days by performing Cell Titer-Glo 2.0 Assay. Germinal center B-cell (GCB)–like DLBCL cell line: Su-DHL10; multiple myeloma cell line: U266; follicular lymphoma (FL) cell line: DOHH2; MCL cell line: MINO; chronic myeloid leukemia (CML) cell line: K562; and acute myeloid leukemia (AML) cell line: MOLM13. Experiments were performed in duplicates. All data are expressed as the mean ± SEM. (B) Immunoblotting analysis of BTK degradation and CRBN neosubstrates after treatment with UBX-382 and thalidomide. Hematological cell lines (U2932, TMD-8, DOHH2, MINO, K562, and WSU-WM) were treated with 100 nM thalidomide and UBX-382 for 24 hours and 72 hours. Immunoblotting was performed to verify the levels of BTK, Ikaros, Helios, Aiolos, GSPT1, and CK1-α using specific antibodies. (C) CRBN neosubstrate protein levels were measured by immunoblotting and values of the remaining substrates, Ikaros, Aiolos, GSPT1, and CK1-α, were normalized using GAPDH intensity value as a loading control. WM, Waldenström macroglobulinemia.