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Materials and methods
RNA isolation. DNA-free total RNA was isolated from untreated and 6-day NGF-treated PC12 cells using the RNAqueous kit (Applied Biosystems, Austin, TX). Poly(A) RNA was prepared using the Poly(A) Purist MAG kit (Applied Biosystems) and poly(A) RNA from rat adult whole Crenolanib was obtained from Applied Biosystems.
Primer extension analysis. Transcription start sites were mapped by primer extension assay using a primer ( Table S1) complementary to the genomic rat sequence (Accession No. NW_047454). Primer and poly(A) mRNA (1 μg) were denatured and annealed at 55 °C for 30 min. First strand cDNA synthesis was carried out using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA) for 50 min at 50 °C as described in [18].
5′ Rapid amplification of cDNA ends. Transcription start sites were mapped by 5′ rapid amplification of cDNA ends (5′RACE) using the GeneRacer kit (Invitrogen) according to the manufacturer’s recommendations and as described in [18]. First strand cDNA synthesis was carried out at 50 °C for 1 h using Superscript III Reverse Transcriptase and gene-specific primers GSP-Bcl-w ( Table S1). Reactions were analyzed on agarose gel and purified fragments were cloned into the pCR4-TOPO vector (Invitrogen) and sequenced (Veritas, Inc., Rockville, MD).