Figure 3.

NGL-2 interacts with PAR complex by means of PAR6. A–C, Colocalization of NGL-2 and PAR complex in hippocampal neurons. A–C, Hippocampal neurons at stage 3 were double stained with anti-NGL-2 and anti-PAR3 (A), anti-PAR6 (B), or anti-aPKCζ (C) antibodies. Arrowhead indicates the tip of the axon. Scale bar, 25 μm. D–I, Interaction between endogeneous NGL-2 and PAR complex proteins in rat brain; homogenates (500 μg of protein) of E18 rat brains were incubated with antibodies against PAR3 (D), PAR6 (F), aPKCζ (H), or NGL-2 (E, G, I). Control IgGs (con IgG) were used as controls. Resulting immunocomplexes were subjected to immunoblotting with indicated antibodies. Five percent of lysates (25 μg of protein) were loaded as input. J, Interaction between NGL-2 and PAR6, PAR3, or aPKCζ proteins in HEK 293 cells. HEK 293 cell lysates expressing the indicated proteins were incubated with anti-Flag antibody. The bound proteins and coimmunoprecipitates were analyzed by immunoblotting with anti-Myc antibody. K, Formation of NGL-2/PAR6/PAR3 ternary complex. HEK 293 cells were cotransfected with Flag-NGL-2 and Myc-PAR3 with or without HA-PAR6. Immunoprecipitates with anti-Flag antibody were subjected to immunoblotting with anti-Myc antibody. Lysates were also probed for the expression of Myc-PAR3, Flag-NGL-2, and HA-PAR6. L, Formation of NGL-2/PAR6/aPKCζ ternary complex in HEK 293 cells. HEK 293 cells were cotransfected with Flag-NGL-2 and Myc-aPKCζ with or without HA-PAR6. Immunoprecipitates with anti-Flag antibody were subjected to immunoblotting with anti-Myc antibody. Lysates were also probed for the expression of Myc-aPKCζ, Flag-NGL-2, and HA-PAR6.