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. 2018 Mar 20;293(18):6762–6775. doi: 10.1074/jbc.RA117.001163

Figure 2.

Figure 2.

VHH6 promotes IL-6 trans-signaling and IL-6·sIL-6R complex formation. A, schematic illustration of VHH6 binding to IL-6·sIL-6R complexes. B, recombinant human IL-6 (0.1–1 ng/ml) and sIL-6R (50 ng/ml) plus increasing amounts of VHH6 (0.1 or 0.5 μg/ml) were mixed and the formed IL-6·sIL-6R complexes were quantified by IL-6·sIL-6R ELISA. Combined data were from three experiments. Error bars represent S.D. C, cellular proliferation of Ba/F3–gp130 cells. VHH6 (0.01–30 μg/ml) was titrated into a setup in which equal numbers of cells were cultured for 3 days without stimulus, with 10 ng/ml of Hyper-IL-6 (HIL-6; control representing 100% trans-signaling complex) or with a combination of 10 ng/ml of IL-6 and 10 ng/ml of sIL-6R. Proliferation was measured using the colorimetric CellTiter-Blue Cell Viability Assay. One representative experiment of three is shown. Error bars represent the S.D. D, cellular proliferation of Ba/F3-gp130 cells. IL-6 (0.01–10 ng/ml) was titrated into a setup in which equal numbers of cells were cultured for 3 days with sIL-6R (10 ng/ml) or with a combination of sIL-6R (10 ng/ml) and VHH6 (10 μg/ml) or HIL-6 alone (10 ng/ml). Proliferation was measured using the colorimetric CellTiter-Blue Cell Viability Assay. One representative experiment of three is shown. Error bars represent the S.D. E, cellular proliferation of Ba/F3-gp130-IL-6R cells. Equal numbers of cells were cultured for 3 days in the presence of HIL-6 (10 ng/ml) or VHH6 (10 μg/ml) and increasing concentrations of IL-6 (0.005–10 ng/ml). Proliferation was measured using the colorimetric CellTiter-Blue Cell Viability Assay. One representative experiment of three is shown. Error bars represent the S.D. F, analysis of STAT3 activation following trans-signaling. Ba/F3–gp130 cells were washed three times, starved, and stimulated with the indicated amounts of HIL-6, IL-6, sIL-6R, and VHH6 for 10 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS gels, followed by immunoblotting using antibodies against phospho-STAT3 or STAT3 (loading control, same samples were used on separated gels). Western blotting data show one representative experiment of three. G, analysis of STAT3 activation following classic signaling. Ba/F3–gp130–IL-6R cells were washed three times, starved, and stimulated with the indicated amounts of IL-6 and VHH6 for 10 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS gels, followed by immunoblotting using antibodies against phospho-STAT3 or STAT3 (loading control, same samples were used on separated gels). Western blotting data show one representative experiment of three.