Ramos (ATCC, CRL-1596), Mino (ATCC, CRL-3000) or K562 (NCI-60) cell lines were counted and diluted to 10 6 cell/ml, using 1 mL per well in a 24-well plate, and U2OS (ATCC HTB-96) cells over expressing human BTK WT or C481S mutant were grown using 2 mL in a 6-well plate (see supplementary methods). Cells were incubated with 1% DMSO or compound in indicated concentrations for 24 hours unless indicated differently or cells were left untreated. Lysates were prepared as previously described , and samples were measured for total protein quantification by Bicinchoninic Acid (BCA) assay (#23225 ThermoFisher Scientific). supplemented with 4x loading buffer including 20 mM DTT, heated to 70°C for 10 minutes and loaded into 4% SDS-PAGE gel, run for 45 minutes at 140 mV, then transferred into nitrocellulose membrane (Biorad) using Trans-Blot Turbo transfer system (Biorad).