Figure 3.

Tomo1 serves as catalytic substrate for neural activity-sensitive Cdk5. A₁, Representative immunofluorescent images of Synapsin 1 (cyan, top) and phosphorylated Cdk5 (pCdk5; heat scale, bottom) in straightened axon segments from neurons treated (30 min) with CSA (50 μm), Rosco (100 μm), or control (DMSO, 0.5%). A₂, Averaged pCdk5 immunofluorescence intensity relative to vehicle control. A₃, Cumulative frequency of pCdk5 intensity across boutons shows that inhibition of Cdk5 by Rosco significantly reduces pCdk5 immunoreactivity, whereas CSA treatment enhances pCdk5 relative to control. B₁–B₃, pCdk5 immunofluorescence images comparing chronic silencing of neural activity by TTX (24 h, 1 μm) with control. Averaged pCdk5 intensity (B₂) and cumulative frequency of pCdk5 (B₃) intensity demonstrate that chronic dampening of neural activity significantly reduces Cdk5 activation. A, B, Analysis was performed on 45 images/preparation per condition and repeated on 3 neuronal preparations. C, Immunoblots showing coprecipitation of Tomo1 and Cdk5 from cultured hippocampal neuron lysates regardless of primary target of IP (Tomo1 or Cdk5). D, In vitro phosphorylation of affinity-purified Tomo1 by Cdk5/p25 as measured by ³²P labeling. ³²P-radioactive signal (top) and anti-Tomo1 immunoreactivity (bottom) on Western blot corresponding to the following conditions: absence of Cdk5/p25 kinase (H₂O), Cdk5/p25 kinase (Cdk5), and Cdk5/p25 + 1 mm Rosco (Cdk5 + Rosco). *p < 0.05.