Fig. 4.

Met interacts with Fis1 and triggers its tyrosine phosphorylation. a The schematic diagram of proteomic analysis of Met co-binding proteins. b The comma blue stain of proteins co-IP with Met or normal IgG antibody. c Fis1 was detected in Met co-binding beads determined by MS analysis. d, e Huh7 cells were stimulated with HGF (100 ng/ml, 20 min) and then applied to IP assay with anti-Met antibody (d) or anti-Fis1 antibody (e). f, g Huh7 cells were treated with crizotinib (1 µM, 1 h) or ARQ-197 (5 µM, 1 h) and then applied to IP assay with anti-Met antibody (f) or anti-Fis1 antibody (g). h Schematic diagram of Fis1 truncations. The deleted regions are represented by lines. i HEK293T cells were transiently co-transfected with the indicated plasmids for 48 h. Cells were lysed and immunoprecipitated with anti-Flag antibody. Co-immunoprecipitated HA-tagged Met was detected by immunoblotting. j Purified GST–Fis1 fusion protein was incubated with recombinant activated Met kinase for 30 min in the kinase buffer with ATP, and then subjected to WB. k Purified GST–Fis1 fusion protein was incubated with activated Met kinase in the presence of crizotinib or protein–tyrosine phosphatase (PTP1B) for 1 h, and the phosphorylation levels of Fis1 were detected with WB. l Purified His–Fis1 fusion protein was incubated with activated Met for 30 min, and then subjected to LC–MS analysis to detect phosphorylated tyrosine sites. m Purified Fis1 (WT), Fis1 (Y38F), Fis1 (Y87F), and Fis1 (Y38/87F) fusion proteins were incubated with activated Met for 30 min. Two kinds of anti-phosphorylation antibodies (p-Tyr-1000 and p-Tyr 4G10) detecting total phosphotyrosine levels were used in WB. n His–Fis1 fusion protein was incubated with Met kinase for 30 min in the presence of crizotinib (1 µM, 1 h) or ARQ-197 (5 µM, 1 h) or not, and the phosphorylation levels of Fis1 were determined with specific Fis1 pY38 antibody. o Huh7 cells were treated with crizotinib (1 µM) or ARQ-197 (5 µM) for 1 h, and the expression levels of p-Fis1 (Y38) was determined with specific Fis1 pY38 antibody