Figure 6.

CD44, Met, and HGF form a multimeric complex. (A) Western blots with anti-CD44 antibodies or with avidin of immunoprecipitates (IP) from HT29 cells treated with HGF where indicated, and cross-linked with DTSSP (see Materials and Methods; multimeric complex precipitations were also obtained without prior cross-linking; data not shown). For preclearing [precl(HGF)], lysates were immunoprecipitated with anti-HGF antibodies, and the supernatant was again immunoprecipitated with anti-HGFα. Rabbit IgG is a control antibody. (B) Western blots (5G8 antibodies) of immunoprecipitates from ASv6 cells (7.5% gel) and immunoprecipitates from MDCK cells using avidin for detection of biotinylated HGF (10% gel). (C) Immunoprecipitates of ASv6 or AS lysates using Met-specific antibodies were resolved under nonreducing conditions by SDS-PAGE (10%). A single band was identified that stained with either avidin (shown here) or antibodies directed against Met or CD44 (data not shown). The band was excised from the gel, incubated with dithiothreitol and rerun on SDS-PAGE. Western blots with avidin, 5G8 (αCD44), and α Met are shown. (D) Western blot for Met expression in lysates of ASv6 or AS cells. (E) Same procedure as in A. For immunoprecipitations, IM7 or control antibodies were used. Where indicated, cells were incubated with BIWA antibodies or IM7 at 37°C for 5 min prior to induction with biotinylated HGF.