Fig. 2

Overexpression of NISCH in primary cortical neurons affects dendritic spine morphogenesis. A Confocal images of whole neurons transfected with control or NISCH OE construct. Scale bars represent 20 μm. Dendritic branches were from each corresponding neuron, and scale bars represent 5 μm. Neuronal morphologies were visualized by staining for enhanced green fluorescent protein (EGFP). B Dendritic spine densities in the neurons transfected with control or NISCH OE construct. Results are presented as mean ± SEM (control, n = 20; NISCH OE, n = 20), and P < 0.05 after multiple corrections were defined as significant. All neuronal experiments were replicated at least twice with consistent conduct and acquisition parameters, and within each experiment, the dendritic spines were counted for each condition from more than three separate cultures. C Nischarin interacts with Actr2. pCDH-CMV-NISCH-FLAG-EF1-mCherry + Puro and the pCAC-Actr2-HA plasmid co-transfected into HEK293T cells. The cells were lysed for the immunoprecipitation (IP) experiment with an anti-HA antibody and the immunoblotting (IB) experiment with an anti-FLAG antibody after 72 h of transfection; 2% of the cell lysates were used as the input sample (top panel). Nischarin-FLAG and Actr2-HA were overexpressed in HEK293T cells (bottom panel). D Western blot analysis of Psd95 in HT22 cells overexpressing Nischarin. NISCH overexpression plasmid or control plasmid was transfected into HT22 cells for 72 h. Gapdh is used as an internal control. *P < 0.05, ***P < 0.0001. NISCH OE, NISCH overexpressed