Confirmation of the activity of the IRES element by western blot analysis. A) Schematic representation of the Di-cis vectors with EGFP as second cistron. (I) indicates pRE as control vector, (II) pREΔCMV with the corresponding promoterless control vector, (III) shows the pRE-IR2 with IRES element IR2 (nt631 to nt990) in the inter-cistronic region, and (IV) the corresponding promoterless construct (pRE-IR2ΔCMV). B) Western blot of the expression product of the Di-cis constructs transiently transfected in N2A cells: The blots show the increased expression of the downstream EGFP cistron mediated by the IR2 element (III). C) Western blot of β-actin as loading control.