Functional analysis of the long (pRF-L) and short (pRF-S) CIRP 5′-UTRs by transient transfection. The long and short 5′-UTRs of CIRP mRNA were (A) cloned into the pRF bicistronic expression vector and (B) cotransfected into NIH-3T3 with a control β-galactosidase plasmid. The relative firefly to renilla luciferase expression is shown after normalization to control β-galactosidase expression. The pRF vector was used as a negative control, while the pRMF vector containing the cmyc IRES in the intercistronic region was used as a positive control. Transfected cells were grown under either normothermic (37°C, gray) or hypothermic (32°C, black) growth conditions and assayed for luciferase activity 24 h post-transfection. For further details see the text.
