Sxbp1

Lungs from each group of mice were homogenized in 500 μl lysis buffer (10 mM Tris-HCL pH 7.5, 120 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 1% Triton X-100) containing protease and phosphatase inhibitor cocktails (Roche) using a sonicator on ice. Samples were centrifuged at 14,000 g for 20 min at 4 °C, and the supernatants were collected. Sample protein concentrations were measured using the BCA protein assay kit (Pierce Biotechnology, Rockford, IL), and 65-μg protein samples were separated by electrophoresis on 4–12% gradient Bis-Tris gels and transferred to nitrocellulose membranes. After blocking with 0.1% casein, membranes were incubated with primary antibodies against BiP, pIRE1α, CHOP, cleaved caspase-12 (Cell signaling Technology, Danvers, MA), sXBP1, NF-κ p65 (Santa Cruz Biotechnology, Santa Cruz, CA), 4-HNE (Abcam, Cambridge, MA), or β-actin (Sigma-Aldrich, St. Louis, MO). 4-HNE is stable, making its quantification more reliable than direct quantification of ROS
After washing, membranes were incubated with the appropriate fluorescent-conjugated secondary antibodies. Bands were detected using an Odyssey FC Imaging system (LI-COR, Lincoln, NE). Band intensities were quantified with densitometry and represented as fold changes relative to controls. The corrected band intensity data from two Western blots was used to plot each histogram.

Primary antibodies targeting the following proteins were used in this study: p-eIF2α, IRE-1α, CHOP (Cell Signaling Technologies, Danvers, MA, USA), GRP78, ATF6α, PERK, p-PERK, sXBP-1, β-actin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), p-IRE-1α (Abcam, Cambridge, MA, USA) and monoclonal PDI (clone 1D3, Enzo Life Sciences, Farmingdale, NY, USA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology. IC87114 was obtained from Calbiochem (San Diego, CA, USA). Ovalbumin, lipopolysaccharide, N-ethylmaleimide (NEM), 4-hydroxy-2-nonenal (4-HNE) and dihydroethidine (DHE) were purchased from Sigma-Aldrich (St Louis, MO, USA).

Rats were quickly decapitated under deep anaesthesia, and the left hemispheres were isolated for Western blot as described before [32 (link)]. The polyvinylidene fluoride (PVDF) membranes were incubated with primary antibodies as follows: TXNIP (1:500; Abcam, Cambridge, Mass), TRX1 (1:2000; Abcam), NLRP3 (1:1000; Abcam), cleaved Caspase-1 (1:500; Abcam), cleaved IL-1β (1:500; Abcam), cleaved Caspase-3 (1:1000; CST, Danvers, MA), BCL-2 (1:1000; CST), sXBP1 (1:100; Santa Cruz), phosphorylated-PERK (p-PERK, 1:200; Santa Cruz), eukaryotic translation initiation factor-2α (eIF2α) (1:100; Santa Cruz), phosphorylated-eIF2α (p-eIF2α, 1:500; Abcam), carbohydrate response element-binding protein (ChREBP) (1:100; Santa Cruz), activating transcription factor 5 (ATF-5) (1:2000; Abcam) and β-actin (NeoBioscience Technology Co., Ltd, Beijing, China). Membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies, visualized with the enhanced chemiluminescent reagent kit (ECL, Engreen Biosystem Co., Ltd. Beijing, China) and analysed using the Fusion system (Fusion fx 7 Spectra, Vilber, France). Results are expressed as a percentage of the values for β-actin.