Additional file 1 of Receptor engineering constitutes feedback control and robustness of IL-23R signaling and highlights importance of intracellular cytokine receptor signaling motifs

Additional figure 1. Analysis of Ba/F3-gp130-IL-12Rβ1-IL-23R cells with regard to receptor and SOCS3 expression. A) Flow cytometry analysis of IL-23 receptors on the surface of Ba/F3-gp130-IL-12Rβ1-IL-23R cells, indicated as solid line. Expression was detected via antibodies against extracellular domains of IL-12Rβ1 (left panel) or IL-23R (right panel). Gray-shade area indicates non-transfected Ba/F3-gp130 cells (negative control). B) Quantification of SOCS3 mRNA expression in stimulated Ba/F3-gp130-IL-12Rβ1-IL-23R cells. The specified times were used for stimulation with HIL-6 (10 ng/ml) or HIL-23 (10 ng/ml). ***p ≤ 0.001, ns not significant. Additional figure 2. SOCS3 interacts with IL-23R. A) Co-IP of FLAG-tagged SOCS3 and full-length IL-23R using ANTI-FLAG® M2 affinity gel. One of two independent experiments is shown. L, lysates; IP, co-immunoprecipitates. B) Co-IP of FLAG-tagged SOCS3 and IL-23R deletion variants using ANTI-FLAG® M2 affinity gel. One of two independent experiments is shown. L, lysates; IP, co-immunoprecipitates. Additional figure 3. IL-23 receptor surface expression. Flow cytometry analysis of IL-23 receptors on the surface of Ba/F3-gp130 cells, indicated as solid line. Expression was detected via antibodies against extracellular domains of IL-12Rβ1 (left panel) or IL-23R (right panel). Gray-shade area indicates non-transfected Ba/F3-gp130 cells (negative control). Additional figure 4. SOCS3 induced negative feedback of Ba/F3-gp130 cell lines stimulated with HIL-6. STAT3 and ERK1/2 activation in Ba/F3-gp130 cells expressing IL-12Rβ1 and IL-23RW395V (A), IL-23RI400V-P401V (B) or IL-23RW395V-I400V-P401V (C) treated with HIL-6 (10 ng/ml) for indicated time points or left untreated. Equal amounts of proteins (50 μg/lane) were analyzed via specific antibodies detecting phospho-STAT3 and STAT3, phospho-ERK1/2 and ERK1/2, and SOCS3. Western blot data shows one representative experiment out of three. Additional figure 5. IL-23 receptor surface expression. Flow cytometry analysis of IL-23 receptors on the surface of Ba/F3-gp130 cells, indicated as solid line. Expression was detected via antibodies against extracellular domains of IL-12Rβ1 (left panel) or IL-23R (right panel). Gray-shade area indicates non-transfected Ba/F3-gp130 cells (negative control). Additional figure 6. Analysis of SOCS3 expression in stimulated Ba/F3-gp130-IL-12Rβ1-IL-23R cells. Quantification of SOCS3 mRNA expression in stimulated Ba/F3-gp130-IL-12Rβ1-IL-23R cells. The specified times were used for stimulation with HIL-6 (10 ng/ml) or HIL-23 (10 ng/ml). **p ≤ 0.01, **p ≤ 0.05, ***p ≤ 0.001, ns not significant. Additional figure 7. IL-23 receptor surface expression. Flow cytometry analysis of IL-23 receptors on the surface of Ba/F3-gp130 cells, indicated as solid line. Expression was detected via antibodies against extracellular domains of IL-12Rβ1 (left panel) or IL-23R (right panel). Gray-shade area indicates non-transfected Ba/F3-gp130 cells (negative control).