Fig. 8 | Nature Communications

Fig. 8

From: Synthetic cytokine receptors transmit biological signals using artificial ligands

Fig. 8

Engineered heterotrimeric SyCyRs based on GVHH-IL-12Rβ1 and GVHH-gp130 homodimerization fail in STAT3 trans-phosphorylation. a Schematic illustration of IL-12Rβ1-SyCyRs simulating STAT3 trans-phosphorylation using 2xGFP-mCherry. GVHH-IL-12Rβ1: GFP-nanobody fused to 15 aa of ECD, TMD, and ICD of IL-12Rβ1, lacking any STAT-binding motifs. CVHH-IL-23R-ΔJAK-B variant: mCherry-nanobody fused to 16 aa of ECD, the TMD and ICD of the IL-23R lacking JAK activation site (ΔJAK-B). b STAT3 activation in indicated Ba/F3-GVHH-IL-12Rβ1 cells lines were stimulated with 16% CHO-K1 conditioned supernatant containing mCherry, 2xmCherry or conditioned supernatant containing 400 ng/ml of the other indicated ligands for 60 min or with HIL-23 (10 ng/ml) for 60 min and HIL-6 (10 ng/ml) for 15 min. Total proteins (25 µg/lane for HIL-6 and 50 µg/lane for other ligands) were analyzed for phospho-STAT3 and STAT3. Western blot data show one representative experiment out of three. c Proliferation of indicated Ba/F3-SyCyR(IL-23/2A) and Ba/F3-GVHH-IL-12Rβ1 cell lines were cultured in the presence of 2xGFP-mCherry (0.1–1600 ng/ml). One representative experiment out of two is shown. Results are mean ± s.d. of three replicates. d Schematic illustration of gp130-SyCyRs simulating STAT3 trans-phosphorylation with 2xGFP-mCherry. GVHH-gp130-ΔSTAT: GFP-nanobody fused to 13 aa of the ECD, the TMD and ICD of gp130 lacking the STAT-binding motifs. CVHH-IL-23R-ΔJAK-B: mCherry-nanobody fused to 16 aa of the ECD, the TMD and ICD of the IL-23R lacking JAK activation site. e STAT3 and ERK1/2 activation in indicated Ba/F3-GVHH-gp130-ΔSTAT cell lines were stimulated with 8% CHO-K1 conditioned supernatant containing mCherry, 2xmCherry or conditioned supernatant containing 200 ng/ml of the other indicated ligands for 60 min or HIL-23 (10 ng/ml) for 60 min or HIL-6 (10 ng/ml) for 15 min. Equal amounts of total protein (for STAT3 analysis 25 µg/lane for HIL-6 and 50 μg/lane for other ligands. For ERK1/2 analysis 12.5 µg/lane for HIL-6 and 75 µg/lane for other ligands) were analyzed for phospho-STAT3/ERK1/2 and STAT3/ERK1/2. Western blot data show one representative experiment out of two. f Proliferation of indicated Ba/F3-GVHH-gp130-ΔSTAT cells lines were cultured in the presence of 2xGFP-mCherry (0.1–1600 ng/ml). One representative experiment out of four is shown. Results are mean ± s.d. of three replicates

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