Overexpression of Nex1 induces expression of several genes associated with neuronal differentiation in the absence of NGF. Western blot analyses comparing the expression of neuronal marker protein using equal amounts of total protein from PC12 cells and the three PC12–Nex1-M(A), PC12–Nex1-M(B), and PC12–Nex1-M(C) clones. The specific antibodies used for this study are described in Materials and Methods. The antigen–antibody complexes were detected by chemiluminescence. Overexpression of Nex1 in untreated cells stimulates the expression of the GAP-43 protein (A), the neuronal specific βIII-tubulin protein (B), the bHLH differentiation factor NeuroD (C), and the mitotic inhibitor p21WAF1 protein (D). As expected, PC12 cells do not express GAP-43 and NeuroD proteins. The p21WAF1 protein and the neuronal-specific βIII-tubulin protein are expressed at much lower levels in PC12 cells.