Degradation and cleavage of p35 and its mutants.

<p>(A) Expression of p35 in COS-7 cells. Wild-type p35 (wt) or its mutants, p35 A108V:L178L (A108V) and p35 L302I, were expressed alone (–) or together with wild-type (wt) CDK5 or kinase-negative (kn) CDK5 D144N in COS-7 cells. p35 was detected by immunoblotting using an anti-myc antibody. Quantification is shown in the lower panel (means ± SEM, <i>n</i> = 3, one-way ANOVA). (B) COS-7 cells expressing p35 wt, A108V or L302I mutant were treated with 50 μg/mL of cycloheximide (CHX) for the indicated times. p35 and its mutants were detected by immunoblotting using an anti-myc antibody. Quantification is shown in the lower panel (means ± SEM, <i>n</i> = 3, one-way ANOVA). (C) Cleavage of p35 to p25 in COS-7 cells. COS-7 cells transfected with p35 and knCDK5 were treated with 20 mM ionomycin for the indicated times. p35 and p25 were detected by immunoblotting using an anti-myc antibody. p25 production is quantified and the ratio of p25/p35 is shown on the right (means ± SEM, <i>n</i> = 3, one-way ANOVA).