Novel AU-rich proximal UTR sequences (APS) enhance CXCL8 synthesis upon the induction of rpS6 phosphorylation
Fig 8
CXCL8 UTR reporter expression is sensitive to the (rpS6 S235/236+phos)/rpS6 ratio.
(A and B) Graphical representations of the CXCL8-5’+3’ Nluc and Cntrl-Nluc mRNAs are shown. These Nluc reporter plasmids were transfected into parallel cell cultures along with plasmids for the expression of TAK1+TAB1, myrAKT, MKK7-JNK2, caPKA or the control empty pcDNA plasmid. In each of these co-transfection experiments, an equimolar ratio of each plasmid species was used. After overnight incubation, the resulting Nluc protein and mRNA expression levels were quantified via luciferase assay and real-time PCR, respectively. The ratio of UTR-Nluc over Cntrl-Nluc expression was then determined and presented as log2 values. Western blots display the expression levels of ERK1/2 T202/204+phos, total ERK1/2, Akt S473+phos, total Akt, rpS6 S235/236+phos, rpS6 S240/244+phos, total rpS6, 4E-BP1 T37/46+phos, total 4E-BP1, eIF4E S209+phos, total eIF4E and β-Tubulin. The band signal intensities were quantified and used to calculate the normalized band intensity ratios as described in the mathematical expressions. The mRNA levels were determined via real-time PCR. Nluc mRNA levels were standardized to the NeoR gene expressed from the pcDNA3.1(+), which is the expression plasmid for the UTR- and Cntrl-Nluc constructs. Each graph symbol (squares or circles) is the result of a replicate experiment. Replicate experiments were performed on different days. For the western blots, two replicates are shown.