Figure 3: DISC1 interferes with ErbB4–PSD95 interaction.

(a) Co-immunoprecipitation (co-IP) of ErbB4 and PSD95 in lysates from mature primary cortical neurons infected with shRNA (control (Cont) or DISC1) and treated with NRG1. Top panels, western blot for PSD95, p-tyrosine and ErbB4 in anti-ErbB4 precipitates; bottom panels, western blot for PSD95 and ErbB4 in input. Cells with DISC1 shRNA showed greater amounts of PSD95 co-precipitating with ErbB4 and greater levels of NRG1-induced tyrosine phosphorylation of ErbB4. (b) Co-IP of ErbB4 and PSD95 in cortical lysates from adult Disc1-LI mice or WT littermates. Top panels, western blot for PSD95, p-tyrosine and ErbB4 in anti-ErbB4 precipitates; bottom panels, western blot for PSD95 and ErbB4 in input. Disc1-LI mice showed greater amounts of PSD95 co-precipitating with ErbB4 and greater levels of NRG1-induced tyrosine phosphorylation of ErbB4. (c) Co-IP of PSD95, DISC1 and ErbB4 in lysates from HEK-293 cells. Cells were transfected with haemagglutinin (HA)-tagged DISC1, ErbB4 and GFP-tagged PSD95 constructs (full length (FL) or with the following deletions: PDZ domain 1 and 2 (ΔPDZ1-2), Src homology 3 domain (ΔSH3) and guanylate kinase-like domain (ΔGK)). Top panels, western blot for HA (DISC1) and ErbB4 in anti-GFP (PSD95) precipitates; bottom panels, western blot for PSD95, ErbB4, DISC1 and GAPDH in input. Amounts of DISC1 and ErbB4 co-precipitating with PSD95-ΔPDZ1-2 were significantly reduced compared with PSD95-FL. Data are represented as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.01. N=3 independent experiments.