Fig. 2

Lj-1-60 is a novel inhibitor targeting Fyn protein kinase. a HEK293T cells transfected with 2 μg and 4 μg of Fyn-flag plasmids for 36 h, it was then harvested and assessed by pull-down assay. Cell lysates incubated with Sepharose coupled with Lj-1-60 were subjected to SDS-PAGE, and analyzed by immunoblotting with antibody anti-flag. b Melanoma cell lysates for pull-down assay was detected by immunoblotting with anti-Fyn antibody. c In vitro kinase assay. A reaction mixture of substrate human recombinant protein Stat3 with myc-tag (676-770 aa), Fyn kinase and Lj-1-60 were incubated at 30 °C for 40 min and subjected to immunoblotting with indicated antibodies. Data are representative of three independent experiments. d Knocked down of Fyn in Sk-Mel-5 and Sk-Mel-28 cell lines by two independent shRNA. Total cell lysates were subjected to immunoblotting using indicated antibodies. Data are representative of three independent experiments. e Lj-1-60 treated in Sk-Mel-5 and Sk-Mel-28 cell lines for 48 h with indicated concentration and analyzed by immunoblot using antibodies p-Stat3(Tyr 705), Stat3, and GAPDH as loading control