Supplementary Figure 5: Biophysical studies to dissect the assembly of TSLP complexes and cellular activity assays to probe observed receptor-receptor contacts at site III.
From: Structural basis of the proinflammatory signaling complex mediated by TSLP
(a) Coomassie-stained SDS-PAGE gel illustrating the purity of the samples for glycosylated TSLP, TSLPR, IL-7Rα and binary TSLP–TSLPR complex used in the binding studies. (b) ITC thermogram for the titration of 4 mM mIL-7Rα with 46 μM mTSLP. (c-d) Recovered ITC-samples from the titration of TSLP with TSLPR (c) and the titration of IL-7Rα with TSLP–TSLPR (d) were analyzed by MALS. (e) Single-cycle kinetics SPR data for the binding of TSLPR to immobilized TSLP. (f) Cellular activity assays employing TSLPR and IL-7Rα mutants to probe receptor-receptor contacts in site III. TSLP-induced STAT5 activity was measured using a luciferace-based reporter system in HEK293T cells expressing wild type and/or mutant forms of either TSLPR or IL-7Rα upon incubation with wild type TSLP. The determined half-maximum effective concentration (EC50) values for STAT5-activation assays were determined as follows: Control experiment with wild type TSLPR and IL-7Rα (EC50= 0.27 nM); IL-7RαH185A (EC50= 0.31 nM); TSLPRQ162A (EC50= 0.28 nM), TSLPRR180S (EC50= 0.38 nM), and TSLPRD177S (EC50= 0.39 nM). Each experiment was carried out in triplicate and error bars were calculated as s.e.m.
