The C and T allelic variants of putative IRES were cloned into an NF-kB-SEAP reporter vector (Takara Bio) using unique HindIII and NruI restriction sites between transcription and translation start sites of the SEAP reporter (Supplementary Material, Fig. S6). Human neuroblastoma BE2C cells (ATCC, Manassas, VA, USA) were grown to 90% confluency in 12- well plates and transiently transfected using Lipofectamine-2000 (Invitrogen) with a mixture of either 0.3 µg of pNF-kB-SEAP-IRES(C/T) constructs or original pNF-kB-SEAP reporter vector and 0.03 µg of pCMC-Luc (CLONTECH) vector per well. The levels of SEAP mRNA and enzymatic activity were determined at 8, 24 and 48 h after transfection, by real-time PCR using SYBRGreen PCR kit (ABI) and by luminometry using a Great Escape kit (Takara Bio). Data were normalized for transfection efficiency by measuring the mRNA levels of Luc for each experimental point, measured by real-time PCR using SYBRGreen PCR kit (ABI). Total RNA was isolated using the RNeasy Mini kit (Qiagen). The isolated RNA was treated with TURBO DNA-free kit (Ambion, Austin, TX, USA) and reverse transcribed by SuperScript III reverse transcriptase (Invitrogen).
