Figure 2

The 3'-UTR of CDK5R1 causes a decrease in luciferase activity. A) Schematic representation of luciferase construct carrying the human 3'-UTR of CDK5R1. The pGL4.71P control construct does not contain CDK5R1 UTR sequences. The Renilla luciferase gene is indicated by the grey bars, and the region of the 3'-UTR is represented by the white bars. The numbers on the right indicate the 3'-UTR nucleotides included in the chimeric construct. Numbering starts from the first nucleotide beyond the stop codon for CDK5R1. Transcription was under the control of the SV40 promoter (hatched bars) and the polyA site (black bars). B) Luciferase activity of pGL4.71P-UTR construct in SK-N-BE, SH-SY5Y, HEK-293 and MCF-7 cell lines assessed using the Dual-Glo Luciferase assay system. Cells were transiently co-transfected with the pGL4.71P-UTR construct (Renilla luciferase) and the pGL3 (Firefly luciferase) vector and were harvested 24 hours post-transfection (see Methods). Luciferase activity of the chimeric reporter gene, normalized for transfection efficiency against the Firefly luciferase activity, is represented as a percentage of the activity observed in cells transfected with pGL4.71P (defined as 100%). Means ± s.d. luciferase values were obtained from at least four independent experiments (* p < 0.01 compared with the corresponding pGL4.71P value). C) mRNA levels of the chimeric transcript. Total RNA was extracted from the cells harvested 24 hours post-transfection, and the reporter gene mRNA levels were analyzed by RealTime PCR as described. mRNA levels of the chimeric reporter gene, normalized against the housekeeping gene GAPDH and for transfection efficiency against the Firefly luciferase mRNA levels, are represented as a percentage of the mRNA levels observed in cells transfected with pGL4.71P (defined as 100%). Means ± s.d. luciferase mRNA values were obtained from at least three independent experiments (* p < 0.01 compared with the corresponding pGL4.71P value).