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. 2004 Mar 15;164(6):851–862. doi: 10.1083/jcb.200309081

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Copyright © 2004, The Rockefeller University Press

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Figure 8. — Overexpression of the L70A Nesca mutant and/or loss of endogenous Nesca expression negatively affects NGF-dependent neurite outgrowth. (A) PC12 cell lines stably expressing the nuclear translocation defective L70A Nesca mutant were assayed for neurite outgrowth (20 ng/ml NGF; 48 h). (B) EGFPNesca expression was silenced in PC12 EGFPNesca D2 cells with the pSilencer vector expressing a siRNA targeted against EGFP. Clones were analyzed for NGF-dependent neurite outgrowth and Nesca expression by Western blotting. Antityrosine hydroxylase antibodies served as a control for protein loading. (C) Endogenous Nesca expression was silenced in stable PC12 cells as described in Materials and methods. Cells were treated for up to five days with 10 ng/ml NGF. Control PC12 cells expressed a scrambled siRNA with no homology to rat mouse or human genomic sequences. Changes in endogenous Nesca expression were determined with an affinity-purified mouse anti-Nesca antibody. Left lane represents murine Nesca expressed in HEK293 cells.