FIGURE 5.
Wnt-5a increases the GDP/GTP exchange of the Gαo subunit, and FZD9 is associated with Gαo in DIV 14 hippocampal neurons. A, hippocampal neurons were stimulated with recombinant Wnt-5a for the indicated lengths of time. The lysates were incubated with specific antibodies that recognize Gαo-GTP or Gαi-GTP. As a positive control, untreated hippocampal neuron lysates were incubated with GTPγS for 90 min at room temperature and then incubated with the antibody. The lysates were analyzed by SDS-PAGE and immunoblotting with antibodies that recognize the total levels of Gαo or Gαi (n = 3). The IgG band shows that equal amounts of the Gαo/i-GTP antibodies were used for immunoprecipitation. Left panel, representative blot images. Right panel, blot quantifications. B, DIV 13 or 14 neurons were treated or not with Wnt-5a (30 min), and the lysates were immunoprecipitated with an anti-Gαo antibody and immunoblotted with anti-FZD9 and anti-Gβ1–5 (this antibody labels all five Gβ isoforms) (n = 4) in the same membrane. Left panel, representative blot images. Right panel, blot quantifications. **, p < 0.01; *, p < 0.05. No Ab, no antibody was added; WB, Western blotting; IP, immunoprecipitation.