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Real-time PCR was performed using Bio-Rad SsoFastTM EvaGreen? Supermix, with the following cycling conditions: GPX4 5 min at 94��C, followed by 45 cycles of 94��C for 30 s, 54.3��C for 30 s and 72��C for 45s. The RNA abundance was expressed as ����Ct, and the fluorescence signals of target gene expression were normalized to that of the internal control (GADPH). Total cellular extracts from RBL-2H3 cells were lyzed in Laemmli buffer. Equal amounts of proteins were loaded, separated by 10% SDS-PAGE before being transferred to polyvinylidene difluoride membranes, and then probed with the following primary antibodies: anti-Orai1 (1:1000 dilution; Abcam, Cambridge, UK), anti-STIM1 (1:1000 dilution; Abcam) and anti-GADPH (1:1500 dilution; Abcam), followed by HRP-conjugated secondary antibody (Abcam). Bound antibodies were visualized using the enhanced chemiluminescence (ECL) detection kit (Invitrogen). Equal loading was confirmed by immunoblotting for GADPH and subjected to SDS-PAGE in an identical manner. Allergen sensitization and challenge was performed as described previously with minor revision (Eisenbarth et al. 2002). The mice were anaesthetized with pentobarbital sodium (60 mg kg?1i.p.) and sensitized intranasally with 100 ��g of ovalbumin (OVA; Sigma) and 1 ��g of LPS (Sigma) in 35 ��l of PBS on days 0, 1, 2 and 3. The sensitized mice were challenged on days 14, 15, 18 and selleck chemical 19 intranasally with 50 ��g of OVA in 35 ��l of PBS under general anaesthesia with pentobarbital sodium (60 mg kg?1i.p.). Methacholine AHR and lung inflammation were evaluated on days 20 and 21, respectively. Mouse peritoneal mast cells were isolated on day 21 for detection of Ca2+ signalling. Twenty-four hours after the final aerosol challenge, AHR was assessed in conscious and unrestrained mice by means of whole-body plethysmography (Buxco, San Diego, CA, USA), as described previously (Albertine et al. 2002; Lee et al. 2012). The enhanced minute pause (Penh) value was evaluated from bronchoconstriction-induced changes in box pressure during expiration and changes in box pressure during inspiration. Mice were exposed to increasing concentrations Osimertinib research buy of nebulized methacholine (from 0 to 100 mg ml?1, dissolved in PBS). The exposure time for each dose was 2 min. The highest Penh value obtained during each methacholine challenge was expressed as a proportion of the basal Penh value seen in response to PBS challenge. Mice were anaesthetized by inhalation of ether and killed by decapitation, and tracheotomies were performed. After ice-cold PBS (0.5 ml) was instilled into the lungs, BALF was obtained by three aspirations via tracheal cannulation. The BALF was centrifuged at 500g, 37��C for 5 min.