Nilotinib

Dasatinib (DASA), imatinib (IMA), nilotinib (NILO), and ponatinib (PONA) were purchased from Cayman Chemical Company (Ann Arbor, USA). After dissolving in DMSO, TKI were kept at – 20 °C until use. Stock concentrations were 20 mM for DASA, IMA and PONA, and 3 mM for NILO, respectively. Peak and IC₅₀ concentrations were chosen according to the literature: DASA 150 nM (peak) and 10 nM (IC₅₀); IMA 3000 nM (peak) and 600 nM (IC₅₀); NILO 3000 nM (peak) and 30 nM (IC₅₀); PONA 145 nM (peak) and 70 nM (IC₅₀) (Peng et al. 2004 (link); Kantarjian et al. 2006 (link), 2010 (link); Rix et al. 2007 (link); Weisberg et al. 2007 (link); Cortes et al. 2012 (link); Menna et al. 2020 (link)). Direct effects of DMSO (maximum level was 0.1% v/v in NILO peak samples) were ruled out by including DMSO only as control throughout all experiments.

LASP1 and CRKL were phosphorylated by recombinant active ABL kinase (BPS Bioscience, San Diego, CA, USA) according to the suppliers recommendations at 30°C in phosphorylation buffer (5 mM Tris, 1 mM MgCl₂, 0.1 mM DTT and 100 μM ATP). For maximal phosphorylation (Supplementary Figure 3), LASP1 had to be incubated with ABL kinase for at least 2h and CRKL for at least 4h (overnight). To block kinase activity in the subsequent experiments, 20fold excess of nilotinib (Cayman Chemical, Ann Arbor, MI, USA) related to ABL kinase concentration was added (Supplementary Figure 3).
Phosphorylation was visualized by Western blot analysis with phosphotyrosine antibody PY20 (1:1000), pCRKL (1:5000) or monoclonal rabbit pLASP1-Y171 specific antibody generated in-house (Supplementary Figure 1) followed by secondary horseradish peroxidase-coupled goat-anti-mouse or goat-anti-rabbit IgG antibody (1:5000) (Biorad, Munich, Germany).