P p38 mapk

The protein samples were extracted from LUAD cells or tumor xenografts via lysing in RIPA buffer (Solarbio). After quantification, equal protein samples were separated by 10% SDS-PAGE, and transferred onto PDVF membrane (Millipore, Danvers, USA). After blocked with skim milk for 2 h, the membranes received another 12 h of incubation with primary antibodies, including anti-CD147, ACOX1, FASN, Rap1, p38 MAPK, p-p38 MAPK, and GAPDH (Abcam). After 2 h of incubation with HRP-conjugated IgG (Abcam), the blots in the membrane were visualized using an ECL kit (Thermo Fisher Scientific) and finally quantified using an Imaging Analysis System (Tanon5200, Shanghai, China).

Antibodies against Atrogin-1 (Cat# ab74023), MuRF-1 (Cat# ab172479), NRF-1 (Cat# ab175932), TFAM (Cat# ab131607), Cytochrome C (Cyt C) (Cat# ab13575), p38 MAPK (Cat# ab197348), p-p38 MAPK (Cat# ab195049), PGC-1α (Cat# ab54481), and GAPDH (Cat# ab9484) were purchased from Abcam (Cambridge, United States). Antibody against COXIV (Cat# bs1533) was purchased from Bioss (Beijing, China). p38 MAPK inhibitor SB203580(4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole) was purchased from Med Chem Express (NJ, United States). Megestrol acetate (Cat# B1377) was purchased from Ape × Bio (Houston, United States).

The lysates of treated cells were reconstituted with loading buffer and run by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Nitrocellulose membranes (Millipore, USA) containing the transferred proteins were soaked in blocking buffer for 2 h and then separately incubated at 4°C overnight with the following primary antibodies: SelS (Sigma, USA), endothelial nitric oxide synthase (eNOS; Proteintech, Wuhan, China), p-c-jun (Proteintech, Wuhan, China), c-jun (Proteintech, Wuhan, China), p-p38 MAPK (Abcam, USA), p38 MAPK (Abcam, USA), inhibitory kappa B α kinase β (IKKβ, CST, USA), p-IKKβ (CST, USA), inhibitory kappa B α (IκBα, CST, USA), p-IκBα (CST, USA), NF-κB p65 (Bioworld, USA), Lamin B (Proteintech, Wuhan, China), and GAPDH (Proteintech, Wuhan, China). After washing for 30 min, the Nitrocellulose membranes were then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The membranes were subsequently treated with enhanced chemiluminescence (ECL, Thermo Scientific, USA) to develop the protein bands, and images were captured using the ChemiDoc MP Imaging System (Bio-Rad, USA). Quantitative analysis of the band intensities was performed with the Quantity One 4.52 software program (Bio-Rad, USA). Lamin B and GAPDH were used as internal controls.

Tumor cells were collected, and intracellular proteins were extracted using RIPA lysate (Solarbio, Beijing, China). The BCA kit was used to determine the protein content of the samples. After protein quantification, protein 70 μg was obtained from each group and added to the loading buffer. The protein was denatured at 95°C for 10 min. Electrophoresis was carried out, the membrane was transferred (Millipore, Bedford, MA, USA), and 5% skim milk was sealed. The following primary antibodies were added: PI3K (Abcam, 1 : 1000), p-PI3K (Abcam, 1 : 1000), AKT (Abcam, 1 : 1000), p-AKT (Abcam, 1 : 1000), ChK2 (Abcam, 1 : 1000), p38MAPK (Abcam, 1 : 1000), p-p38MAPK (Abcam, 1 : 1000), Bax (Abcam, 1 : 1000), Bcl-2 (Abcam, 1 : 1000), Cleaved Caspase 3 (Abcam, 1 : 1000), and GAPDH (Abcam, 1 : 1000), and the samples were incubated at 4°C overnight. The secondary antibody (horseradish peroxidase-labeled sheep anti-rabbit, Abcam, 1 : 5000) was added and incubated at room temperature for 2 h. The images were then taken with a gel imaging system after a chemical illuminator was added. The relative protein content was represented by the target protein GAPDH.

Proteins from cultured neutrophils were prepared with a protein extraction solution (containing 20 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton, 0.1% SDS and 1% protease inhibitor cocktail). Protein concentrations were determined with a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher, Waltham, MA, USA). Then, 40 μg samples of protein extracts were fractionated on 12% polyacrylamide-sodium dodecyl sulfate (SDS) gels and then transferred to nitrocellulose membranes. The membranes were blocked with 5% (w/v) fat-free milk in Tris-buffered saline (TBS) containing 0.05% Tween-20, followed by incubation with primary antibodies to phosphorylated-p38 MAPK (p-p38 MAPK), phosphorylated Mcl-1 (p-Mcl-1), Akt, phosphorylated P65 (p-P65), phosphorylated IκBα (p-IκBα), and α-tubulin (all at 1∶1000; Abcam, Cambridge, UK) at 4°C overnight. The next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1∶5000, Abcam), and bands were visualized with an enhanced chemiluminescence system with short exposure to X-ray films (Kodak, Rochester, NY, USA).

Total protein samples were obtained using lysis buffer (Solarbio, Beijing, China) supplemented with 1% protease inhibitor (Solarbio). After centrifugation at 4°C for 30 min, Bicinchoninic Acid Protein Assay kits (Thermo Fisher Scientific) were applied to examine protein concentrations in the light of specifications. Nuclear and cytoplasmic proteins were obtained with the help of the Nuclear Protein Extraction Kit (Solaibio) in accordance with the manufacturer's descriptions. Then, protein samples were loaded to 10% SDS-polyacrylamide gel and submitted to electrophoresis and transformation to polyvinylidene difluoride membranes (PVDF; Millipore, Billerica, MA, USA). Next, the membranes were probed with first antibodies overnight at 4°C after being blocked in 5% nonfat milk for 1 hour at room temperature: anti-β-actin antibody (1 : 5000 dilution; cat. no. ab8226, Abcam, MA, USA), p-p38 MAPK (1 : 1000 dilution; cat. no. #4511, Cell Signaling Technology, MA, USA), and anti-MAPK1 antibody (1 : 3000 dilution; cat. no. ab182453, Abcam). Then, the membranes were probed with the HRP-conjugated secondary antibodies at room temperature for 1 hour. Following incubation with ECL reagent (Millipore, USA), the protein signaling was measured by using ProfiBlot 48 (Tecan, Switzerland) and quantified by using ImageJ v2.1.4.7 (National Institutes of Health, Bethesda, MD, USA).