Western blotting was performed as described elsewhere [40 (link)]. Briefly, a PVDF membrane was incubated with 5% bovine serum albumin for 2 h, and then incubated overnight at 4 °C with primary antibodies. After washing 3 times, the membrane was incubated for 1 h with species-specific horseradish peroxidase-conjugated antibodies, and developed with ECL detection reagent (ImmunoStar, Wako Pure Chemical). For detection of Met tyrosine phosphorylation, cell lysate was subjected to immunoprecipitation with mouse anti-Met (code:#3127, clone 25H2) antibody (Cell Signaling Technology, Beverly, MA) and phospho-Met was detected using rabbit anti-phospho-Met (Y1234/Y1235) (code:#3077, clone D26) antibody (1:1000 dilution, Cell Signaling Technology). Immune complexes were recovered with Protein G-Sepharose beads (Zymed Laboratories, South San Francisco, CA). For exosome analysis, antibodies against Met (25H2), Rab5 (rabbit, code:#5347, clone C8B1) (1:1000 dilution, Cell Signaling Technology), TRP2 (rabbit, code: BS3320, clone K89, 1:1000 dilution, Bioworld, Louis Park, MN), HSP70 and HSP90 (mouse, code: SPA-810-D and ADI-SPA-830, clone C92F3A-5 and AC88, 1:1000 dilution, Enzo Life Sciences, Farmingdale, NY), and VLA4 (rat, code: ab25247, clone PS/2, 1:1000 dilution, Abcam, Cambridge, MA, USA) were used. Can Get Signal® (TOYOBO, Osaka, Japan) was used for the dilution of antibodies.
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