Fig. 10. Inhibition of MAP kinase affects the distribution of normal and constitutively phosphorylated NF-H within axonal neurites equally. (A) Immunoprecipitation with anti-NF polyclonal antibody R39 from cultures transfected 24 hours previously with GFP-Hwt (wt) or GFP-Hasp (Asp) and probed with anti-GFP antibody; note the presence of GFP-immunoreactive species migrating at 200 kDa. (B) Representative images of cells transfected with GFP-Hwt or GFP-Hasp as indicated, before and after 2 hours' treatment with PD98059; images before and after treatment are of different cells. (C) Densitometric analysis of the ratio of GFP fluorescence in the distal versus the central third of axonal neurites from 10-20 individual cells from two independent experiments before and after a 2-hour treatment with PD98059. The lower graph shows the percentage reduction in GFP signal in the distal third of axonal neurites for each construct following a 2-hour treatment with PD98059, obtained by dividing the ratio before to that after treatment. Consistent with prior reports (see text), the ratio of GFP signal in distal/central axons is lower in cells transfected with GFP-Hasp compared to GFP-Hwt; however, note that 2 hours' treatment with PD98059 reduces this ratio by the same percentage for both constructs. A similar impression is obtained by visual inspection of the cells in panel B. (D) Sequentially captured images of representative axonal neurites of a cell transfected with GFP-Hasp at 15-second intervals commencing 15 minutes after addition of PD98059. The merged image (M) was prepared from the 0 and 30 second images as described in the legend for Fig. 7. Note that many structures in the merged image do not superimpose indicating continued net translocation following the addition of PD98059. (E) The percentage of filamentous or particulate GFP-tagged structures exhibiting net anterograde, retrograde or lack of net movement prior to (dark-shaded bars) and following (pale-shaded bars) a 15-minute treatment with PD98059 from 10-20 individual axons from two independent experiments for each construct quantified as described in Materials and Methods and in the legend to Fig. 7. Images of cells transfected with GFP-Hwt and of cells transfected with GFP-Hasp prior to the addition of PD98059 are not presented because, as indicated by quantification of particles (E) they are essentially identical to those presented for GFP-M in Fig. 7.