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Fig. 6. Manipulation of MAP kinase activity does not alter neurite length or alter steady state levels of actin and tubulin, but does alter C-terminal NF phosphorylation within axonal neuritis. (A) Fluorescent images of axonal neurites of NB2a/d1 cells following labeling with rhodamine-conjugated phalloidin to label actin (Actin) or an anti-tubulin antibody (Tubulin). The accompanying graph shows the relative density of actin and tubulin fluorescence along axonal neurites ±2 hours' treatment with PD98059 determined for 25-50 cells (mean±s.d.); note that there is no significant difference in the presence or absence of PD98059. (B) Cells double-labeled with an antibody against NF-L and the phospho-dependent anti-NF antibody RT97. The graph shows the ratio of RT97 density/NF-L density (as indices of phospho-NFs and total NFs, respectively) before and after a 2-hour treatment with PD98059 (mean±s.d.). Treatment with PD98059 induced a specific reduction in phospho-NFs. (C) The length (mean±s.d.) of 25-50 axonal neurites in duplicate cultures following treatment with PD98059 or transfection with constitutively active (Const-Act) or dominant-negative (Dom-neg) MAP kinase. Axonal neurite length was not altered by manipulation of MAP kinase activity.

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