Figure 2
Synthetic IL-35 receptor complexes consisting of IL-12Rβ2 and gp130 are biologically active. (A) Schematic overview of IL-35-type signaling by IL-12-induced receptor activation of IL-12Rβ1EXR-gp130IR and IL-12Rβ2. (B) Representative histograms of IL-12Rβ1EXR-gp130IR (upper panel) and IL-12Rβ2 (lower panel) surface expression of Ba/F3-gp130/IL-12Rβ1EXR-gp130IR/IL-12Rβ2 cells (light solid lines). Gray-shaded areas indicate Ba/F3-gp130 cells (negative control). (C) Analysis of STAT1/3 and Erk1/2 activation. Ba/F3-gp130/IL-12Rβ1EXR-gp130IR and Ba/F3-gp130/IL-12Rβ1EXR-gp130IR/IL-12Rβ2 cells were washed three times, starved, and stimulated with 4 ng/ml HIL-12 for 30 min or 10 ng/ml HIL-6. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS gels, followed by immunoblotting using specific antibodies for phospho-STAT1/3/Erk1/2 and STAT1/3/Erk1/2. Western blot data show one representative experiment out of three. (D) Cellular proliferation of Ba/F3-gp130/IL-12Rβ1EXR-gp130IR and Ba/F3-gp130/IL-12Rβ1EXR-gp130IR/IL-12Rβ2 cells. Equal numbers of cells were cultured for 3 days in the presence of 4 ng/ml HIL-12. Proliferation was measured using the colorimetric CellTiter-Blue Cell Viability Assay. HIL-6–induced proliferation (10 ng/ml) was set to 100%. One representative experiment out of three is shown. Error bars represent SD. Statistical analysis used a Welch t test (n = 3; *p ≤ 0.05; ***p ≤ 0.001). (E) Analysis of STAT3 target gene expression of Pim-1 in Ba/F3-gp130/IL-12Rβ1EXR-gp130IR/IL-12Rβ2 cells stimulated with 4 ng/ml IL-12 for 2 h. One representative experiment out of two is shown. (F) Schematic overview of IL-35-type signaling by IL-12-induced receptor activation of IL-12Rβ1EXR-gp130IR and IL-12Rβ2EXR-gp130IR. (G) Representative histograms of IL-12Rβ1EXR-gp130IR (upper panel) and IL-12Rβ2EXR-gp130IR (lower panel) surface expression of Ba/F3-gp130/IL-12Rβ1EXR-gp130IR/IL-12Rβ2EXR-gp130IR cells (light solid lines). Gray-shaded areas indicate Ba/F3-gp130 cells (negative control). (H) Analysis of STAT1/3 and Erk1/2 activation of Ba/F3-gp130/IL-12Rβ1EXR-gp130IR, Ba/F3-gp130/IL-12Rβ2EXR-gp130IR and Ba/F3-gp130/IL-12Rβ1EXR-gp130IR/IL-12Rβ2EXR-gp130IR cells as described in (C). Western blot data show one representative experiment out of three. (I) Cellular proliferation of Ba/F3-gp130/IL-12Rβ1EXR-gp130IR, Ba/F3-gp130/IL-12Rβ2EXR-gp130IR and Ba/F3-gp130/IL-12Rβ1EXR-gp130IR/IL-12Rβ2EXR-gp130IR cells as described in (D). One representative experiment out of three is shown. Error bars represent SD. Statistical analysis used a Welch t test (n = 3; **p ≤ 0.01; ***p ≤ 0.001). (J) Analysis of STAT3 target gene expression of Pim-1 in Ba/F3-gp130/IL-12Rβ1EXR-gp130IR/IL-12Rβ2EXR-gp130IR cells stimulated with 4 ng/ml IL-12 for 2 h. One representative experiment out of two is shown. (K) Schematic overview of IL-35-type signaling by IL-12-induced receptor activation of IL-12Rβ1EXR-IL-12Rβ2IR and IL-12Rβ2. (L) Representative histograms of IL-12Rβ1EXR-IL-12Rβ2IR (upper panel) and IL-12Rβ2 (lower panel) surface expression of Ba/F3-gp130/IL-12Rβ1EXR-IL-12Rβ2IR/IL-12Rβ2 cells (light solid lines). Gray-shaded areas indicate Ba/F3-gp130 cells (negative control). (M) Analysis of STAT1/3 and Erk1/2 activation of Ba/F3-gp130/IL-12Rβ1EXR-IL-12Rβ2IR and Ba/F3-gp130/IL-12Rβ1EXR-IL-12Rβ2IR/IL-12Rβ2 cells as described in (C). Western blot data show one representative experiment out of three. (N) Cellular proliferation of Ba/F3-gp130/IL-12Rβ1EXR-IL-12Rβ2IR and Ba/F3-gp130/IL-12Rβ1EXR-IL-12Rβ2IR/IL-12Rβ2 cells as described in (D). One representative experiment out of three is shown. Error bars represent SD. Statistical analysis used a Welch t test (n = 3; ns = not significant; *p ≤ 0.05; **p ≤ 0.01). (O) Cellular proliferation of Ba/F3-gp130/IL-12Rβ1EXR-IL-12Rβ2IR/IL-12Rβ2 and Ba/F3-gp130/IL-12Rβ1/IL-12Rβ2 cells. Equal numbers of cells were cultured for 3 days in the presence of HIL-12 (0.01 to 500 ng/ml). Proliferation was measured using the colorimetric CellTiter-Blue Cell Viability Assay. HIL-6–induced proliferation (10 ng/ml) was set to 100%. One representative experiment out of two is shown. Error bars represent SD. (P) Analysis of STAT3 target gene expression of Pim-1 in Ba/F3-gp130/IL-12Rβ1EXR-IL-12Rβ2IR/IL-12Rβ2 cells stimulated with 4 ng/ml IL-12 for 2 h. One representative experiment out of two is shown.
