RNA extraction and real-time polymerase chain reaction
Total RNA was extracted with TRI-reagent from control or U50,488H treated κ-Neuro-2A cells according to manufacturer’s instructions. Total RNA (1 μg) was used as template for cDNA synthesis using SuperScript II reverse transcriptase (Thermo Fisher). The following primers were designed for Real Time-PCR: Becn1,(forward):5’-GGCCAATAAGATGGGTCTGA-3’; (reverse) 5’-GCTGCACACAGTCCAGAAAA-3’; for ATG5, 5’ (forward) AAGTCTGTCC-TTCCGCAGTC-3’; (reverse) GAAGAAAGTTATCTGGGTAGCTCA-3’; forGAPDH ( forward) 5’-TGTGTCCGTCGTGGATCTGA-3’, (reverse) 5’-CCTGCTTCACCACCTTCT-TGA-3’, using a ΜΧ3000P QPCR System (Stratagene, La Jolla, CA, USA). The expression of the mRNAs was calculated using the ΔCt method.