Enhanced chemiluminescence western blot kit

Proteins were extracted from cells using radioimmunoprecipitation assay buffer (Sigma-Aldrich, St Louis, MO, USA), and protein concentration was quantified using the BCA Protein Assay Kit (Beyotime Biotechnology, Haimen, People’s Republic of China). Equal amounts of proteins (50 µg) from each sample were separated by 10% sodium dodecyl sulfate–polyacrylamide gel and then transferred to a polyvinylidene fluoride membrane (Millipore, Boston, MA, USA). The membrane was blocked by 5% skimmed milk for 1 hour at 37°C. The target proteins were probed with primary anti-Nrg1 (ab180808, 1:1,000), anti-cyclin D1 (ab134175, 1:1,000), anti-N-cadherin (ab18203, 1:800), anti-Twist (ab49254, 1:1,000), anti-MMP2 (ab37150; 1:1,200), and anti-GAPDH (ab9485, 1:2,500) antibodies and incubated at 4°C overnight. Thereafter, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000; Beyotime Biotechnology) at 25°C for 1 hour. Immunoreactive protein bands were visualized using the enhanced chemiluminescence Western blot kit (Thermo Fisher Scientific). Protein band intensity was quantified using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). Relative protein expression was obtained by normalization against GAPDH.

Total protein was extracted from pericytes using radio immunoprecipitation assay reagent (Beyotime Biotechnology, Shanghai, China) containing 100 μg/mL phenylmethylsulfonyl fluoride (Solarbio) and quantified using a bicinchoninic acid protein assay kit (Solarbio). Protein samples were denatured, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene fluoride membrane (EMD Millipore Corp., Burlington, MA, USA). The membranes were then blocked with a blocking solution (QuickBlock, Beyotime Biotechnology) at room temperature for 30 minutes, and then incubated with primary antibody against pro-caspase-1 (1:1000, rabbit, Cat# ab179515, Abcam), Nod1 (1:500, mouse, Cat#sc-398696, Santa Cruz), IL-1β (1:500, rabbit, Cat# ab9722, Abcam), CD9 (1:2000, rabbit, Cat# ab92726, Abcam), CD81 (1:5000, rabbit, Cat# ab109201, Abcam), CD63 (1:500, mouse, Cat#sc-5275, Santa Cruz) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH; mouse, 1:1,0000, Cat# AC033, ABclonal, Wuhan, China) at 4°C overnight. The membranes were incubated with secondary goat anti-rabbit or mouse horseradish peroxidase-linked antibody (1:1000, Abbkine, Cat# A21220 or Cat# A25012) at room temperature for 1 hour. Finally, target bands were visualized using an enhanced chemiluminescence western blot kit (Thermo Fisher Scientific) and semi-quantified using ImageJ software.

At 24 hours after the TBI challenge, the lung samples were homogenized in protein extract solution and protease inhibitors (Beyotime Biotechnology, Shanghai, China) and centrifuged at 12 000 rpm for 10 minutes at 4°C. The total protein concentration was evaluated by the BCA protein assay kit (Thermo Scientific, IL, USA). Equal amounts of total protein were loaded per well on a 12% sodium dodecyl sulfate-polyacrylamide gel and transferred into a polyvinylidene difluoride membrane. The membranes were blocked with 5% BSA for 2 hours at room temperature. Then the membranes were incubated with appropriate dilution of specific primary antibodies (anti-RAGE 1: 1000 and anti-GAPDH 1: 1000) for 12 hours at 4°C. Then the membranes were incubated with the horseradish peroxidase-linked secondary antibody (1: 1000; Beyotime Biotechnology, Shanghai, China). Protein bands were demonstrated by enhanced chemiluminescence Western blot kit (Thermo Scientific, IL, USA). Signals were densitometrically assessed and normalized to GAPDH signals.