Expression of PRL-3 mRNA and protein after cytokine stimulation. (A) Quantification of PRL-3 mRNA by real-time RT-PCR was performed after stimulation of IH-1 and OH-2 cells either with or without IL-6 and IL-21 for 24 hours. The ΔCt value between PRL-3 and β-actin from the unstimulated cells reflects the expression of PRL-3 relative to its internal control β-actin. The ΔCt value for the unstimulated IH-1 cells was 2.68 with a standard variation of 0.17 and for the unstimulated OH-2 cells it was 22.49 with a standard variation of 0.31. Thus, standard variation for all ΔCt values from RT-PCR reactions was between 1% and 2% of its ΔCt value. The relative expression level of PRL-3 to β-actin in the unstimulated cells was represented by 2^−ΔCt and was arbitrarily set to 1. The relative expression of PRL-3 to β-actin in the stimulated cell culture, 2^−ΔCt, was then normalized to that of the unstimulated cell culture, 2^{−ΔCt(unstim)} / 2^{−ΔCt(stim)}, and is illustrated on the y-axis. (B) PRL-3 expression was determined by Western analysis after an 18-hour incubation with various cytokines. Cytokine concentrations were as follows: IL-6, 5 ng/mL; IL-21, 20 ng/mL; IL-15, 20 ng/mL; TNF, 10 ng/mL; HGF, 150 ng/mL; IGF-1, 100 ng/mL, and SDF-1, 75 ng/mL. The loading control was GAPDH (bottom panels).