Bicinchoninic acid protein assay kit

Following treatment, A549 cells were harvested and lysed using radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.) supplemented with PMSF (1 mM). The whole cell lysates were centrifuged at 12,000 × g for 15 min at 4°C, and protein content in the supernatant was determined by the bicinchoninic acid protein assay kit (Applygen Technologies, Inc.). Proteins samples (30 µg per lane) were separated by 10% SDS-PAGE and transferred to PVDF membranes at 4°C for 90 min. After blocking with 5% non-fat milk at room temperature for 60 min, membranes were incubated with the following primary antibodies overnight at 4°C: Anti-PI3K p85α (cat. no., ab191606; 1:1,000); anti-phosphorylated (p)-PI3K p85α (Y607; cat. no., ab182651; 1:1,000); anti-AKT1/2/3 (cat. no., ab179463; 1:10,000); anti-p-AKT1 (Ser473; cat. no., ab81283; 1:1,000); and anti-GAPDH (cat. no., ab181602; 1:10,000). All primary antibodies were purchased from Abcam. The membranes were then probed with IgG-horseradish peroxidase-conjugated goat anti-rabbit (cat. no., ab2057; Abcam; 1:10,000) and goat anti-mouse (cat. no., ab6789; Abcam; 1:10,000) secondary antibodies for 1 h at room temperature. Finally, the proteins were detected using ECL Enhanced Chemiluminescent kit (Thermo Fisher Scientific, Inc.). Densitometric analysis was performed using ImageJ 1.44p software (National Institutes of Health).

At 6 h after LPS or corresponding treatment, the mesenteric arteries were separated under an operating microscope, and stored at −75 to −80°C. Subsequently, the vascular tissue was pulverized using a MagNA Lyser automatic tissue homogenizer (Roche Diagnostics International AG, Rotkreuz, Switzerland) with 50 µl cell lysis buffer (Beyotime Biotechnology Research Institute, Shanghai, China), and was centrifuged at 850 × g at 0–4°C for 10 min using the 3–30K high-speed refrigerated Laborzentrifugen (Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany). Phosphorylated (p-)RhoA, p-myosin phosphatase target subunit 1 (Mypt1) and MLCP levels were examined using the mouse-specific ELISA kits (Jiangsu Hope, Inc., Zhenjiang, China; for p-RhoA, HBT-0587M; p-Mypt1, HBT-0684M; and MLCP, HBT-0622M) in accordance with the manufacturer's protocol. Antibodies for p-RhoA (ab41435; 1:2,000), p-Mypt1 (ab59203; 1:4,000), and MLCP (ab59235; 1:4,000) were purchased from Abcam (Cambridge, MA), and used for the ELISA analysis. Protein concentration of the vascular homogenate was measured using a bicinchoninic acid protein assay kit (Applygen Technologies Inc., Beijing, China), according to the manufacturer's instructions, for the normalization of protein levels.

Western blotting was performed to measure the protein expression levels of NF-κB phosphorylated (p)-p65, NF-κB p65, Bax, Bcl-2 and proliferating cell nuclear antigen (PCNA). Total protein was extracted from A549 cells using cold RIPA buffer (Roche Diagnostics) and total protein was quantified using the Bicinchoninic Acid Protein Assay kit (Applygen Technologies, Inc.). Proteins (15 µg/lane) were separated via SDS-PAGE on 10% gels and transferred onto nitrocellulose membranes. The membranes were incubated overnight at 4˚C with primary antibodies which were all purchased from Cell Signaling Technology, Inc. targeted against: Bax (cat. no. 2772; 1:1,000) and Bcl-2 (cat. no. 3498; 1:1,000), PCNA (cat. no. 13110; 1:1,000), NF-κB p-p65 (cat. no. 3033; 1:1,000), NF-κB p65 (cat. no. 8242; 1:1,000) and GAPDH (cat. no. 5174; 1:1,000). Following primary antibody incubation, the membranes were blocked in 5% non-fat milk at room temperature for 1 h and incubated with anti-rabbit IgG secondary antibody (cat. no. 7074, 1:10,000; Cell Signaling Technology, Inc.) at room temperature for 2 h. Protein bands were visualized by enhanced chemiluminescence (Thermo Fisher Scientific, Inc.) using the Odyssey Infrared Imaging system (LI-COR Biosciences). Densitometry was quantified by ImageJ software (version. 1.8.0; National institutes of Health). GAPDH was used as the loading control.

The PAG was quickly removed from coronal slices containing the PAG, which were prepared according to The Rat Brain in Stereotaxic Coordinates (sixth edition) (Paxinos and Watson, 2007 ). Total protein was extracted by homogenizing the PAG tissue in ice-cold radio-immunoprecipitation assay lysis buffer, and protein content was measured using bicinchoninic acid protein assay kit (Applygen Technologies Inc., Beijing, China). Protein samples were separated by electrophoresis on SDS polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were washed with TBS/T (Tris-buffered saline mixed with 0.05% Tween-20) containing 5% nonfat dry milk for 1 h to block nonspecific antibody binding sites, incubated with antibodies against Hsp70 protein (R&D, Minneapolis, Minnesota, USA) or β-actin (Santa Cruz, California, USA) at 4°C overnight and then treated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz, California, USA) for 1 h at room temperature. The blots were probed by chemiluminescent detection method (Applygen Technologies Inc. Beijing, China) and then exposed to X-ray films. The content of the Hsp70 protein in blots was quantified by a Gel Doc 2000 densitometer (Bio-Rad, Hercules, California, USA) and normalized to signals of β-actin protein.