Bicinchoninic acid protein assay reagent

Total protein was extracted with ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (CWBIO) containing phenylmethylsulfonyl fluoride (PMSF) and a phosphatase inhibitor (48:1:1) according to the manufacturer's instructions. Protein levels were quantified using the bicinchoninic acid protein assay reagent (Beyotime, China). Fifteen micrograms of protein was added to each well, separated by 10%/15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with skim milk for 1 h at room temperature, followed by incubation with a primary antibody at 4°C on a rocker overnight. Primary antibodies against BMP2 (Proteintech, 18933-1-AP, USA), COL1 (Proteintech, 14695-1-AP), C/EBPα (Santa Cruz, sc-365318, China), FABP4 (Santa Cruz, sc-271529 SAMPLE), P62 (Abcam, ab109012, USA), LC3B (Abcam, ab192890), and Beclin 1 (Abcam, ab207612) were used. After incubation with secondary antibodies for 1 h at room temperature, a BeyoECL Plus Kit (Beyotime) was used for detection. Gray value analysis was used to analyze the Western blotting results; the target protein gray value was divided by the corresponding internal reference gray value to obtain the relative protein expression.

PC12 cell lysates were extracted using a BCA Protein Assay Reagent kit (Beyotime Institute of Biotechnology, Shanghai, China), following mimic or inhibitor treatment. Protein concentration was determined using the bicinchoninic acid protein assay reagent (Beyotime Institute of Biotechnology). Equal quantities of protein from each sample were loaded and electrophoresed on 10% SDS-PAGE gels (Beyotime Institute of Biotechnology), and transferred to nitrocellulose membranes [Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China]. After blocking with non-fat dried milk, membranes were probed with polyclonal rabbit anti-human acetyl-p53 [dilution, 1:1,000 (cat. no. CS2525); Cell Signaling Technology, Inc., Danvers, MA, USA], polyclonal rabbit anti-human Bcl-2 [dilution, 1:1,000 (cat. no. BS4023); Bioworld Technology, Inc., St. Louis Park, MN, USA] or polyclonal rabbit anti-human SIRT1 [dilution, 1:1,000 (cat. no. CS2327); Cell Signaling Technology, Inc.] antibodies. Antibody signals were visualized using a Chemiluminescent Detection kit, according to the manufacturer's instructions (Beyotime Institute of Biotechnology). Experiments with blank and negative controls were conducted in parallel. The relative band intensities of the blots were quantified with Adobe Photoshop software (Adobe Systems, Inc., San Jose, CA, USA)..

Total proteins of NHBE cells were extracted using a protein extraction kit (NE-PER™ Nuclear and Cytoplasmic Extraction Reagents; Pierce, Thermo Fisher Scientific, Inc.) and the protein concentration was measured using bicinchoninic acid protein assay reagent (Beyotime Institute of Biotechnology). A 10% SDS-PAGE gel was prepared with 20 µg protein samples loaded in each lane. The samples were mixed with the loading buffer and boiled at 100˚C for 5 min. The protein on the gel was then transferred to a nitrocellulose membranes and blocked with 5% skimmed milk powder at room temperature 1 h. GAPDH primary antibody (cat. no. ab70699; 1:5,000; Abcam) was chosen as the internal reference. The membranes were first probed with the anti-TLR4 mouse polyclonal antibody incubated at 4˚C overnight (cat. no. sc-293072; dilution 1:1,500; Santa Cruz Biotechnology, Inc.) and then with a corresponding horseradish peroxidase-conjugated secondary antibody (cat. no. sc-525409; 1:5,000; Santa Cruz Biotechnology, Inc.) at room temperature for 1.5 h. An enhanced chemiluminescence kit (EMD Millipore) was used for visualization of the results and quantification of the bands was performed using the Image J software (v1.8.0; National Institutes of Health).

RAW 264.7 cells were induced with RANKL and M-CSF to generate multinucleated osteoclasts. Additionally, RAW264.7 cells were treated with vehicle or DADS (80 μg/ml) for 2 h followed by stimulation with the indicated concentration of RANKL for 0, 15, 30, or 60 min. Cells were lysed in a lysis buffer containing 10 mM Tris, pH 7.2, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% Triton X-100, and 1% deoxycholic acid supplemented with protease and phosphatase inhibitor cocktail. All of the protein concentrations were assessed and subsequently adjusted to 40 μg/μl using the bicinchoninic acid protein assay reagent (Beyotime Biotechnology). For Western blots, 40 μg of protein samples were subjected to SDS-PAGE followed by transfer onto PVDF membranes. After blocking in 5% skim milk for 2 h, they were incubated with rabbit primary antibodies at the following dilutions overnight at 4°C: NFATc1, 1:1000; c-Fos, 1:1000; NF-κB p65, 1:1000; p–NF-κB p65, 1:1000; IκB-α, 1:1000; p–IκB-α, 1:1000; STAT3, 1:1000; p-STAT3 Ser727, 1:1000; p-STAT3 Tyr705, 1:1000; β-actin, 1:1000. Next, they were incubated with secondary antibodies (1:2000) at room temperature for 1 h. Finally, protein bands were visualized by exposing in ChemiDoc XRS+ Imaging System (Bio-Rad) and analyzed by ImageJ software. β-Actin served as an internal control.