Fig. 5.

cdk5 and p25 abnormalities in thenpc-1 −/− mouse brain. A, cdk5/p35 immunoblots. Whole-brain lysates containing 40 μg of protein from +/+ and −/− mice were resolved on 12% gels and blotted with the indicated antibodies. cdk5 levels remained unchanged in −/− mice compared with controls. The p35 C-terminal antibody showed no difference in p35 levels but an enrichment of the 25 kDa p35 fragment (p25) in −/− mice (left panel, mouse). Similar preparations containing 100 μg of protein from a human control and an NPC hippocampus were immunoblotted with cdk5 and p35C antibodies. cdk5 and p35 levels were invariant, but p25 was elevated in NPC (right panel, human).B, cdk5 IP/kinase activity. Whole-brain lysates (left panel) from +/+ and −/− mice were immuno- precipitated with cdk5 (C-8) antibody. Immunoprecipitate (cdk5 IP) was immunoblotted with a different cdk5 antibody to show that the relative recoveries of enzyme in the samples were similar (Cdk5). Catalytic aliquots of cdk5 immunoprecipitate were incubated with kinase buffer containing histone H1 and [γ-³²P]-ATP for 20 min at 30°C, and the reaction mixtures were resolved on 12% gels. The incorporation of [γ-³²P]ATP into histone H1 (H1) was significantly higher with cdk5 IP from −/− mice compared with +/+ mice (left panel), on the basis of equivalent H1 loading demonstrated by staining of the gel with Coomassie blue (gel). The cdk5 activity in 5- to 10-week-old −/− mice was increased 1.5-fold over that seen in +/+ mice (p < 0.01; n = 12;left panel). Right panel, The increase in cdk5 activity in the forebrain (Forebr.) and brainstem (B.stem) but not in the cerebellum (Cblm.) of a 5-week-old −/− mouse compared with those of a 5-week-old +/+ mouse (all scales, ×1000). C, P-GSK-3β immunoblots. Whole-brain lysates containing 50 μg of protein from +/+ and −/− mice were resolved on 10% gels and blotted with P-GSK-3β antibody (P-GSK-3) or primary sequence-dependent antibody (GSK-3). The intensities of the resulting bands were quantitated densitometrically, and no significant differences (p = 0.11) were observed between the npc-1 and control mice.