FIGURE 3.

Long and short isoforms of IL-31Rα exhibit similar signaling competences. A, luciferase reporter gene assay. HeLa cells were transiently transfected with a STAT3 luciferase reporter and expression plasmids encoding either the short isoform 3 or long isoform 1 of human IL-31Rα. As a control, an equal amount of the empty vector (pCDH) was co-transfected. One day post-transfection, cells were treated with rhIL-31 at rising concentrations as indicated. Luciferase activity was assessed after 24 h of cytokine stimulation. Data represent mean values of three independent experiments carried out in duplicate. Error bars indicate standard deviations. B and C, IL-31-dependent CCL2/MCP-1 expression by transfected HeLa cells. Ccl2/Mcp1 mRNA expression was analyzed after 4 h of IL-31 stimulation by qRT-PCR (B), and CCL2/MCP-1 secretion was measured by ELISA 24 h post IL-31 induction. Data represent mean values of two independent experiments carried out in duplicate. Error bars indicate standard deviations. D, Western blotting. HeLa cells transiently transfected with IL-31Rα isoforms 3 and 1 in pCDH expression vector were treated for 15 min with rhIL-31 at the indicated concentrations. Tyrosine phosphorylation of STAT3, STAT1, STAT5, and ERK1/2 was detected by specific antibodies. To control for equal loading, nonphosphorylated protein was detected. One out of three independent experiments is shown. E, HeLa cells transiently transfected with IL-31Rα isoforms 3 and 1 in pcDNA-His expression vector were treated for 15 min with the indicated concentrations of rhIL-31, and phosphorylation of STAT3, STAT1, STAT5, and ERK1/2 was detected by specific antibodies. To control for equal loading, nonphosphorylated protein and β-actin is shown. IL-31Rα expression was monitored by a His tag-specific antibody. One representative experiment is shown.