Platelets of dasatinib or nilotinib treated CML patients were purified by gel filtration [17 (link)] that was performed on Sepharose CL-2B column. In the in vitro experiments, control platelets were first gel filtrated and then were pretreated by dasatinib/nilotinib and subsequently activated by convulxin.
Gel filtrated platelets (4 × 107 from each sample) were lysed with lysis buffer containing 1% TritonX-100 in PBS supplemented with a cocktail of tyrosine phosphatase inhibitor from Sigma (St. Louis, MO, USA). Platelet lysates were separated by polyacrylamide gel electrophoresis and subjected to western blotting. Specific phosphorylation of Lyn, Fyn, and Src kinases were visualized, using phosphospecific antibodies (Ab) and biotinylated secondary Ab followed by avidin-biotin complex for 30 min. Bands were demonstrated by enhanced chemiluminescence (ECL).
Gel filtrated platelets (4 × 107 from each sample) were lysed with lysis buffer containing 1% TritonX-100 in PBS supplemented with a cocktail of tyrosine phosphatase inhibitor from Sigma (St. Louis, MO, USA). Platelet lysates were separated by polyacrylamide gel electrophoresis and subjected to western blotting. Specific phosphorylation of Lyn, Fyn, and Src kinases were visualized, using phosphospecific antibodies (Ab) and biotinylated secondary Ab followed by avidin-biotin complex for 30 min. Bands were demonstrated by enhanced chemiluminescence (ECL).