Figure 2. Coculture of neurons with Notch ligand-overexpressing cells.

(a) Schematic depiction of the coculture of neurons and NIH3T3 cells. 5 or 6 DIV neurons were cocultured with Notch ligand-expressing NIH3T3 cells for 24 hours and subjected to the analyses. (b) Immunoblot analysis of primary neurons cocultured with NIH3T3 cells expressing either EGFP or Jagged1 with antibodies against Notch1 intracellular domain (upper), cleaved Notch1 (middle) and Jagged1 (bottom). The gels have been run under the same experimental conditions, and representative blots are cropped and used in this figure. Full-length gels are presented in Supplementary Fig. S1. (c) Immunoblot analysis of the cocultured neurons with antibodies against several synaptic membrane proteins. syp, synaptophysin 1; syn, synapsin 1; syt, synaptotagmin 1; βIIItub, neuron-specific β-III-tubulin. The gels have been run under the same experimental conditions, and representative blots are cropped and used in this figure. (d) Densitometric analysis of synaptophysin 1 levels in neurons cocultured with NIH3T3 cells expressing EGFP, Jagged1 (Jag1), Delta-like1 (Dll1) or Neuroligin 1 (NLG1). The band intensities of synaptophysin 1 were normalized to β-III-tubulin for each sample (n = 5–17, means ± SEM, **P < 0.01, ***P < 0.001, compared with EGFP by Steel-Dwass test). (e) Schematic depiction of co-culture without direct cell-cell contact where neurons on the coverslips and neuron cocultured with NIH3T3 cells at the bottom of culture dish. (f) Immunoblot analysis of lysates of neurons on coverslip shown in (e). Jag1-3T3 did not affect the protein levels of synaptophysin 1 and VGLUT1 in neurons on the coverslips, which do not interact with NIH3T3 cells in the same conditioned media of the coculture. The gels have been run under the same experimental conditions, and representative blots are cropped and used in this figure.