Fig. 1
From: Synthetic cytokine receptors transmit biological signals using artificial ligands
SyCyRs for IL-23 (SyCyR(IL-23/2A)) simulate IL-23-induced signal transduction. a Schematic illustration of IL-23 SyCyR and GFP-mCherry fusion protein. GVHH-IL-12Rβ1: GVHH fused to 15 aa of the ECD, TMD, and ICD of IL-12Rβ1. CVHH-IL-23R: CVHH fused to 17 aa of ECD, TMD, and ICD of IL-23R. b Proliferation of Ba/F3-gp130 cell lines with HIL-6 and GFP:mCherry cultured with synthetic ligands (6.25 ng/ml) or HIL-6 (10 ng/ml). Stimulation with mCherry was made with the same volume as with GFP (0.25%). One representative experiment out of three is shown. c Proliferation of Ba/F3-SyCyR(IL-23/2A) and Ba/F3-gp130 cells with GFP-mCherry and Ba/F3-IL-12Rβ1-IL-23R cells with HIL-23 cultured in the presence of ligands (0.1 to 1600 ng/ml). One representative experiment out of five is shown. d Proliferation of Ba/F3-SyCyR(IL-23/2A) and Ba/F3-gp130 cells with GFP-mCherry fusion protein in the presence of GVHH-CVHH. Cells were cultured in the presence of GFP-mCherry (GC; 10 ng/ml) and increasing concentrations of GVHH-CVHH (0.1–100 ng/ml). One representative experiment out of four is shown. Results in b, c, and d are mean ± s.d. of three replicates. e JAK activation in Ba/F3-gp130, Ba/F3-IL-12Rβ1-IL-23R, and Ba/F3-SyCyR(IL-23/2A) cells stimulated with 100 ng/ml of indicated synthetic ligands or HIL-6 (100 ng/ml) for 20 min. Equal amounts of total protein (50 μg/lane) were analyzed using specific antibodies for phospho-JAK2 and JAK2. Western blot data show one representative experiment out of three. f STAT3, ERK1/2, and AKT activation in Ba/F3-SyCyR(IL-23/2A) and Ba/F3-gp130 cells treated with 100 ng/ml of the indicated ligands or HIL-6 (10 ng/ml) for 30 min. Stimulation with mCherry was made with the same volume as with GFP (2%). Equal amounts of total protein (50 μg/lane) were analyzed using specific antibodies for phospho-STAT3/ERK1/2/AKT and STAT3/ERK1/2/AKT. Western blot data show one representative experiment out of three. g STAT3 activation in U4C cells expressing SyCyR receptors were stimulated with 100 ng/ml of the indicated synthetic ligands or HIL-6 (10 ng/ml) for 60 min. Equal amounts of total protein (25 μg/lane for HIL-6 and 50 µg/lane for other ligands) were analyzed using specific antibodies for phospho-STAT3 and STAT3. Western blot data show one representative experiment out of two
