(a) Recombinant PlGF-induced increase in IL-17 secretion from T cells. Splenic CD4+ T cells (1 × 106) from WT mice maintained in conventional facilities were stimulated with α-CD3/28 (1 μg/ml) plus recombinant PlGF (rPlGF; Sino Biological Inc., Wayne, PA, USA) in the absence or presence of the anti-PlGF antibody (α-PlGF Ab, 50 μ/ml) for 48 hours. IL-17 levels in the culture supernatants were measured using an ELISA. *P < 0.05, †P < 0.05 compared with PlGF 200 ng/ml alone. (b) Increase in p-STAT3 levels by recombinant PlGF. Splenic CD4+ T cells were incubated with recombinant PlGF (200 ng/ml, Sino Biological Inc.) in the absence or presence of α-PlGF Ab (50 μ/ml) for 1 hour. The levels of p-STAT3 were assessed by immunoblotting. (c) Upregulation of Rorc expression by recombinant PlGF. Sorted CD4+ T cells were stimulated with α-CD3/28 plus recombinant PlGF (Sino Biological Inc.) for 12 hours. Levels of the Rorc mRNA were determined by quantitative real-time PCR. Gapdh mRNA was used as an internal control. Fold inductions were calculated using the 2-ΔΔCt method. * P < 0.05. (d) Production of PlGF-2-Fc in HEK-293T cells. The Fc-fused PlGF-2-overexpressing vectors for the production of PlGF-2-Fc in HEK-293T cells were constructed. Mouse or human Fc-fused PlGF-2 was produced using transient gene expression with serum-free suspended HEK-293T cells (see the Methods section). Purified Fc-tagged PlGF-2 protein was analyzed on BoltTM 4–12% Bis-Tris Plus Gels under nonreduced and reduced conditions. Lane 1: non-reduced homodimeric form of human Fc-fused PlGF-2 (arrow), lane 2: reduced monomeric form of human Fc-fused PlGF-2 (arrowhead), lane 3: non-reduced homodimeric form of mouse Fc-fused PlGF-2 (arrow), and lane 4: reduced monomeric form of mouse Fc-fused PlGF-2 (arrowhead). (e and f) Increase in IL-17 production and Rorc expression in CD4+ T cells by the PlGF-2 that we isolated and purified from -overexperssed HEK-293T cells, determined by ELISA and quantitative real-time PCR, respectively. * P < 0.05.