Cooperative effect of p54^nrb and hnRNPM on FGF1 IRES activation.

<p>(A) Schema of the constructs used for C2C12 cell co-transfection. The target plasmid is the bicistronic dual luciferase vector with the FGF1 promoter and IRES. The effector plasmids express p54^nrb (p54) or hnRNPM (HM), or co-express p54^nrb and hnRNPM. The latter plasmid is a bicistronic contruct containing the FGF1 IRES. (B) Western blots of transfected proliferating C2C12 cell extracts using antibodies against p54(αHM), hnRNPM (αHM) of GAPDH as a control (αGAPDH). (C-E) Luciferase activities measurement of co-transfected proliferating or differentiating C2C12 cell extracts. LucR activity reflects the FGF1 mRNA promoter activity (as cap-dependent translation does not significantly vary, as shown by RNA transfection, see Table B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136466#pone.0136466.s004" target="_blank">S4 File</a>) (C). LucF reflects IRES-dependent translation but is also dependent on mRNA amount (D). LucF/LucR ratio reflects the IRES activity normalized to mRNA amount, expressed relatively to the control (co-transfection with empty vector) (E). Experiments were performed in biological triplicates and repeated three times. The statistical test used is the Student test. (mean +- standard deviation, *p<0.05, **p<0.01, ***<0.001).