Figure 4.

Phosphorylation of the α₄ nAChR subunit in synaptosomes. Phospho-antibodies were raised to the PKCε phosphorylation sites S465 and S467 in the human α₄ subunit, corresponding to S468 and S470 in mouse. (A) The level of phosphorylated S465 to non-phosphorylated S465 was not significantly different between wild-type and Prkce^−/− mouse synaptosomes. (B) The S467 phospho-antibody detected a strong band from HEKtsA201 cells expressing native α₄β₂ nAChRs ‘S’ and did not detect a band from cells expressing the S467A mutation ‘A’. The non-phospho-α₄ antibody detected bands from both native and S467A mutant receptors. Corresponding GAPDH levels are shown in the lower panel. The S467 phospho-antibody detected several non-specific bands in an overexposure from HEKtsA201 cells that do not express α₄β₂ nAChRs ‘E’. (C) The phospho-α₄ S467 and non-phospho-α₄ S467 antibodies detected bands in synaptosome preparations from wild-type and Prkce^−/− mouse brains. The corresponding GAPDH blots are shown below each panel. The 60 kDa molecular weight band was quantified from the phospho-α₄ S467 blots. Molecular weights are indicated on the side (kDa). (D) The level of phosphorylated α₄ S467 to non-phosphorylated α₄ S467 was 13% higher in synaptosomes from wild-type compared with Prkce^−/− mouse brains. *P < 0.05 compared with wild-type mice, n = 6 mice per genotype.