Fig. 1.

Depolarization promotes NCAM E18 skipping in new transcripts independently of protein synthesis and of CaMKIV inhibition. N2a cells were treated with different concentrations (A) or 60 mM KCl (B, C, and E–H) for 16 h. (D) Rat cultured hippocampal neurons were treated with 40 mM KCl for 20 h or left untreated. After these treatments, cells were harvested, total RNA prepared, and alternative splicing for the indicated genes was assessed with specific primers by a 30-cycle radioactive RT-sqPCR (A–C and G) or real-time RT-qPCR (D–F and H) as described in Materials and Methods. In A, half the cells were harvested and the other half were incubated in normal medium for another 20 h (Recovery). Treatments in E–H were performed in the presence or absence of the transcription inhibitor actinomycin D (Act D; 5 μg/mL), the protein inhibitor CHX (5 μg/mL), or the CaMKIV inhibitor KN93 (20 μM) as indicated. (CPEB4, cytoplasmic polyadenylation element binding protein 4; NR1, NMDA receptor 1; Ctl., control; Dep., depolarization.) Bars show means ± SD corresponding to 2 or 3 independent experiments.