Adenosine diphosphate (adp)

N-terminal 6His tagged recombinant human ALK expressed in baculovirus Sf21 was purchased from EMD Millipore (Billerica, MA, USA) (purity ≥60% by sodium dodecyl sulfate polyacrylamide gel electrophoresis) and aliquoted to 1 μL fractions when it was used the first time (to avoid multiple freeze/thaws for subsequent experiments). BNP7787 was prepared by a proprietary method (purity >97%, no mesna was detected by mass spectroscopy). Kinase inhibitor, PF02341066 (crizotinib), was purchased from Selleck Chemicals, LLC. (Houston TX, USA). Polyglutamate-tyrosine (PolyGT) substrate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Kinase assay buffer was prepared and consisted of 20 mM HEPES, 0.1% Brij 96, 10 mM NaF, 1 mM Na₃VO₄, and 10 mM MnCl₂ adjusted to a final pH of 7.5. Half-area 96-well microplates were purchased directly from Corning Incorporated (Corning, NY, USA). ADP-Glo reagents were purchased from Promega (Madison WI, USA) and consisted of ADP, ATP, ADP-Glo, kinase detection reagent buffer, and kinase detection substrate. All other reagents were purchased from Sigma-Aldrich Co (St Louis, MO, USA). A Tecan Ultra microplate reader with XFluor software (V4.51; Tecan [Morrisville, NC, USA]) and RdrOle software (V4.50; Tecan) were used in this study.