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. 2013 May 27;8(5):e63967. doi: 10.1371/journal.pone.0063967

Figure 4. Over-expression of EBI3 blocks IL-12p70 production in CHO cells.

Figure 4

A. Biscistronic IL-12p40 and p35 expression were compared before being introduced into a CHO cell by transfection. Two clones (C1 and C2) with comparable levels of IL-12p40 and IL-12p70 as detected by ELISA were selected. B. The biological activity of IL-12 was detected by inducing IFNγ production in C57/black mouse CD4+ T cells. C. The EBI3 expression plasmid was transfected into IL-12p70 expression clone 1 to detect EBI3 expression by Western blotting. Three clones (C1–1, C1–2 and C1–3) with high levels of EBI3 expression plus a non-transfected IL-12p70 expression clone (C1) were used to detect EBI3 expression by Western blotting. The culture supernatants from these 4 clones, plus wild type CHO cells were harvested for use in the IL-12p70 ELISA. D. The culture supernatants were also used to detect IL-35 (EBI3 and p35) heterodimeric protein secretion by immune precipitation Western blotting.