Fig. 4. Purified Cdk5-p35 phosphorylates NF subunits in cell-free analyses. (A) Autoradiographic analyses following SDS-gel electrophoresis of Triton-X-100-insoluble cytoskeletons from day 17 embryonic chicken brains incubated for 2 hours with [³²P]orthophosphate with or without Cdk5 and p35. Migratory position of NF-H, NF-M and NF-L are indicated. The accompanying graph presents the relative density of 200 kDa NF-H incubated for 2 hours with [³²P]orthophosphate with or without Cdk5 and p35. Notice the marked increase in radiolabel associated with NF subunits and, in particular, that associated with NF-H following incubation with Cdk5-p35. (B) Immunoblot analyses of 100 µg Triton-X-100-insoluble cytoskeletons incubated for 2 hours at 30°C ±25 µg Cdk5 and 25 µg p35 probed with RT97 and R39 that reacts with all NF subunits regardless of phosphorylation state (Jung et al., 1998) as an index of total NF-H; only the 200 kDa region of immunoblots is presented. The accompanying graph shows the percentage increase in immunoreactivity for each antibody following incubation with Cdk5-p35. Notice the specific increase in phospho-NF (RT97) immunoreactivity.