Protein g sepharose beads

The sources of antibodies and reagents are as followed: anti-pY291 Fas (prepared as previously described¹³ and validated for immunoblotting¹³ and flow cytometry Fig. S8); anti-Src, anti-Yes (R&D Systems); anti-human Fas (C20, for immunoblotting) and anti-Cyclin D1 (Santa Cruz); anti-Fas (Apo1–3, for co-immunoprecipitation) (Enzo); anti-EGFR, anti-pY1068 EGFR, anti-pY705 STAT3, anti-STAT3, anti-pS473 Akt, anti-pT308 Akt, anti-Akt, anti-pErk1/2, and anti-Histone H3 (Cell Signaling Technology); anti-Erk1/2 (Sigma); anti-GAPDH (Calbiochem); horseradish peroxidase conjugated anti-rabbit and anti-mouse (Jackson ImmunoResearch); anti-mouse IgG-Alexa Flour 488 (Life Technology); Recombinant human EGF (Gibco), recombinant amphiregulin (R&D Systems); protein G sepharose beads (Zymed Laboratories); WST-1 (Interchim); Matrigel (BD Bioscience). Lipofectamine RNAiMAX reagent (Invitrogen) was used for siRNA transfection according to the manufacturer’s protocol for reverse transfection.

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.