Synthesis and Characterization of Masitinib and Derivatives

Masitinib (99% pure) and masitinib-NH₂ (99% pure) were provided by AB Science S.A. Masitinib-NH₂-LC-Biotin was synthesised from masitinib-NH₂ (for synthesis and characterisation, see Supplementary Note 1, Supplementary Fig. 14). DI-39 was synthesised as described previously^{39 (link), 40 (link)}. Protein kinase inhibitors were purchased from Selleckchem. Reagents and substrates for kinetic assays were all purchased from Sigma-Aldrich. Each reagent was obtained as powder and dissolved in H₂O or DMSO and stored as aliquots at −20 °C. Fresh dilutions were prepared for each experiment. Primary antibodies used were a rabbit polyclonal anti-deoxycytidine kinase antibody (ab96599, Abcam, 1:1000), a rabbit polyclonal anti-ERK2 antibody (sc-154, Santa Cruz Biotechnology, 1:2000), a rabbit polyclonal anti-AKT1 antibody (#9272, Cell Signalling Technology, 1:1000), a mouse monoclonal anti-Lyn antibody (610004, BD Biosciences, 1:1000), and a rabbit polyclonal anti-Kit antibody (#3074, Cell Signalling Technology, 1:1000). Primary antibodies were detected using 1:20,000 horseradish peroxidase-conjugated anti-rabbit antibody (Jackson Immunoresearch Laboratories Inc.) or 1:20,000 horseradish peroxidase-conjugated anti-mouse antibody (Dako).