Figure 6.

NCAM promotes phosphorylation of the exocyst complex by the FGF receptor. A, Sec8 immunoprecipitates (IP) from NCAM+/+ and NCAM−/− brain lysates probed by Western blot with sec8 and phosphotyrosine antibodies (pY20). Mock immunoprecipitation with nonspecific IgG served as control. Note that tyrosine phosphorylation of sec8 is reduced in NCAM−/− brains. B, Recruitment of exo70 and sec8 from NCAM+/+ cytosol to NCAM+/+ growth cone membranes analyzed by Western blot with exo70 and sec8 antibodies. Membranes were treated with alkali to strip peripheral proteins. Control membranes were incubated with the buffer used for cytosol preparation instead of cytosol. When indicated, the cytosol was preincubated with staurosporine, FGFR inhibitor PD173074, or src family inhibitor PP2. Labeling for L1 served as a loading control. Note that staurosporine (lane 4) and PD173074 (lane 3), but not PP2 (lane 5) inhibit recruitment of exo70 and sec8. Graph shows mean + SEM optical densities from 3 experiments. Levels in inhibitor-treated probes normalized to inhibitor nontreated probes (set to 100%, dashed line) are shown. *p < 0.05 (paired t test, compared to probes nontreated with inhibitors). C, Sec8 immunoprecipitates from NCAM+/+ growth cones analyzed by Western blot with sec8 and pY20 antibodies. Mock immunoprecipitation with nonspecific mouse IgG served as control. Growth cones were treated with NCAM antibodies or nonspecific rabbit IgG in the presence or absence of the FGFR inhibitor. Note that the NCAM antibodies induce tyrosine phosphorylation of sec8 (lane 1), which is blocked by the FGFR inhibitor (lane 3). Graph shows mean + SEM optical densities from three experiments with the signals for nonspecific rabbit IgG-treated growth cones set to 100%. *p < 0.05 (paired t test).