For KCl depolarization of neurons, DIV 6 neurons were quieted overnight in 1 µM TTX and 100 µM APV, and they were incubated for 0 or 6 hours in 55 mM KCl. Total RNA was isolated from cultures using 1 ml Trizol/well according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Isolated RNA was treated with DNAseI Amplification Grade (Invitrogen, Carlsbad, CA) and cDNA library was synthesized by cDNA High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). The cDNA was the source of input for quantitative PCR, using a Step One Plus Real-Time PCR Instrument and SYBR Green reagents (Applied Biosystems, Carlsbad, CA). The relative expression plot was constructed using concentration values that were normalized to corresponding tubulin concentrations.
