Analysis of Ten-4 expression in Neuro-2a neurite outgrowth. A) Quantitative RT-PCR of Ten-4 in Neuro-2a cells during neurite outgrowth. mRNA expression of Ten-4 increased with time after induction of neurite outgrowth. Ten-4 mRNA expression was normalized using GAPDH mRNA expression. Normalized expression of Ten-4 on d 0 was set as 1.0. Error bars = se. B) Western blotting of Ten-4 in Neuro-2a cells during neurite outgrowth. Highest expression of Ten-4 was observed 3 d after induction of neurite outgrowth. Expression of α-tubulin was used as a control. C) Immunostaining of Ten-4 in Neuro-2a cells before induction of differentiation (a–c), 1 d (d–f), and 3 d (g–i) after induction of neurite outgrowth. Ten-4 protein was localized and accumulated at the tips of the neurites. Panels b, c; e, f; and h, i show enlarged images of boxed areas in panels a, d, and g, respectively. Arrows indicate filopodia-like membranous protrusions; arrowheads indicate Ten-4 expression at a growth cone or along a neurite. Scale bar = 10 μm.