Monoclonal anti flag antibody conjugated to hrp

ELISA was performed using Ni-NTA HisSorb plates (Qiagen, Germany). In brief, 100 µL of a solution containing hTSLPR with C-terminal octa-histidine tag (TSLPR-His) was added to each well and incubated for 2 h at room temperature. After incubation, the plate was washed twice with 200 µL of PBS with 0.05% Tween-20 to remove unbound TSLPR-His, and candidate inhibitors as well as TSLP with N-terminal FLAG tag (FLAG-hTSLP) were added at 100 µL each. After overnight (18 h) incubation at 4 °C, the plate was washed twice and blocked with 100 µL of blocking buffer (PBS with 0.05% Tween 20 and 1% nonfat dry milk). The plate was washed twice to remove unbound FLAG-hTSLP and then coated with 100 µL of monoclonal anti-FLAG antibody conjugated to HRP (Sigma-Aldrich Co., USA) for 2 h at room temperature. Following incubation, the plate was washed five times and further treated with 200 µL of o-phenylenediamine dihydrochloride (Sigma-Aldrich Co., USA) solution and incubated for 30 min. After incubation, 1N HCl was added to stop the reaction. Optical densities (ODs) were measured at 450 nm using a microplate spectrophotometer. The TSLP-inhibitory effect was calculated using the following formula: $$\mathrm{Inhibitory}\mathrm{effect}(\%)=(1-\mathrm{OD}\mathrm{of}\mathrm{sample}/\mathrm{OD}\mathrm{of}\mathrm{control})\times 100$$

Intracellular phospho-STAT5 (pSTAT5) was measured using a BD LSR Fortessa Flow cytometer (BD Biosciences, San Diego, CA, USA). NMR spectra were recorded on a Bruker SPECTROSPIN 300 MHz spectrometer (Bruker Corporation, Billerica, MA, USA). For the pSTAT5 assay measuring TSLP inhibitory activity, recombinant human TSLP was purchased from R&D Systems (Minneapolis, MN, USA) and BD Cytofix/Cytoperm and Alexa Fluor 647 mouse anti-STAT5 (pY694) were purchased from BD Biosciences (San Diego, CA, USA). For the ELISA assay to assess hTSLP–hTSLPR interaction, Ni-NTA HisSorb plates were purchased from Qiagen (Hilden, Germany), and monoclonal anti-FLAG antibody conjugated to HRP and o-phenylenediamine dihydrochloride were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Iscove’s modified Dulbecco’s medium (IMDM) and fetal bovine serum (FBS) were purchased from Hyclone Laboratories Inc. (Logan, UT, USA). Penicillin-streptomycin was purchased from Gibco Industries Inc. (Auckland, NZ, USA).