FIGURE 6.

AKAP79 regulates the phosphorylation of Robo3.1 by PKC. A–F, HEK293 cells were transfected with Robo3 and treated with PDBu (A–C) or vehicle (DMSO; D–F) for 10 min. Cells were incubated with a Robo3 antibody and the phospho-PKC substrate antibody, and a PLA reaction was carried out between these two antibodies (B, C, E, and F; orange). Cells were subsequently stained using a V5 antibody to recognize Robo3.1 (A, C, D, and F; green). G, analysis of PLA signal normalized to Robo3 expression (means ± S.E. (error bars), n = 3, >100 cells, p ≤ 0.05, one-sample Student's t test). H, Robo3 and the m₁ muscarinic receptor were coexpressed in HEK293 cells with control or AKAP79 shRNA. Cells were treated with vehicle (H₂O) or 10 μm Oxo-M for 2 min. V5 immunoprecipitations (IP) of cell lysates were immunoblotted with the PKC substrate antibody (top). Robo3.1 expression (top middle), AKAP79 knockdown (bottom middle), and m₁ receptor expression (bottom) were confirmed by immunoblotting. I, quantification of phospho-Robo3 signal present in immunoprecipitations normalized to Robo3 expression (means ± S.E., n = 4, p ≤ 0.05, unpaired Student's t test). A.U., arbitrary units.