Figure 6

C-menin mediates neurite localization of α7 subunit-containing nAChRs. (A,B) ICC characterization of the neurite localization of C-menin and α7-nAChRs in NTC shRNA and MEN1 shRNA lentivirus transduced hippocampal cultures (A; n = 18 images, 5 independent samples each; representative images, DIV 7) or hippocampal cultures maintained in the presence of 0.1% DMSO or 20 μM PD150606 (B; n = 12 images, 2 independent samples each; representative images, DIV 7). Cells were labeled with the menin C-terminal epitope antibody (i-ii-a) or fluorophore-tagged α-BTX (iii-iv-a), and neurites were visualized with GFP (Ai-iv-b) or neurofilament (Bi-iv-b) antibodies, (i-iv-c) shows merged channels. Scale bars, 20 μm. (C,D) Summary data, the α-C-menin signal in neurites was reduced upon MEN1 knockdown (C), which coincides with a reduction of the neurite α-BTX signal (D). (E) Summary data, reduction of C-menin abundance by calpain inhibition (see Fig. 2D) coincides with a reduction of the neurite α-BTX signal (ROIs: n ≥ 36 each; see Table S5), indicating that the synaptic targeting of α7-nAChRs requires the menin C-terminal fragment. Asterisks, statistical significance (Mann-Whitney U test); *P < 0.05. ***P < 0.001.