NF-κB activity assay

An ELISA-based assay was performed to measure endogenous NF-κB activities as described previously [27]. Briefly, after BECs were treated with 20 ng/ml IL-4 for 24 h in the presence or absence of M-βCD or 2-APB (M-βCD or 2-APB was administrated half an hour before IL-4 using), the cells were lysed with lysis buffer (20 mM HEPES pH 7.5, 0.35 M NaCl, 20% glycerol, 1% NP-40, 1 mM MgCl₂·6H₂O, 0.5 mM EDTA, 0.1 mM EGTA) containing a protease inhibitor cocktail (Calbiochem, La Jolla, CA, USA) on ice for 10 min. The supernatant obtained after centrifugation at 14,000 rpm for 30 min at 4 °C was recovered. The double-stranded probe with single-stranded-linker was generated by 1:1 mix of the following two oligonucleotide with the sequences of 5′-AGTTGAGGGGACTTTCCCAGGCC-(C)34-C-3′, the 3′ end biotinylated and 5′-GCCTGGGAAAGTCCCCTCAACT-3′, respectively. The probe was denatured at 94 °C for 10 min, annealed at room temperature overnight and then linked to streptavidin-coated 96-well plates (Roche, Mannheim, Germany) by incubating 2 pmol of probe per well for 1 h at 37 °C in 50 μl PBS. After wash, 20 μl of cell extract were mixed with 30 μl of binding buffer (4 mM HEPES pH 7.5, 100 mM KCl, 8% glycerol, 5 mM DTT, 0.2% BSA, 40 μg/ml salmon sperm DNA) in the above microwells incubated at room temperature with mild agitation (200 rpm) for 1 h. After wash, mouse anti-NF-κB p65 monoclonal antibody (1:1000 diluted) was incubated for 1 h at room temperature. After wash, peroxidase-conjugated goat anti-mouse IgG were incubated at room temperature for 1 h. After wash, 100 μl tetramethylbenzidine was incubated at room temperature for 10 min before adding 100 μl of stopping solution (2 M H₂SO₄). Optical density was then read at 450 nm under a microplate reader (Biotek Instruments, Winooski, VT, USA) using a 655-nm reference wavelength. Backgrounds are determined in lysis buffer and subtracted before data analysis.