Enhanced chemiluminescence ecl kit

Cells were lysed with 1× RIPA buffer (Millipore, Temecula, CA, USA) containing 1 mM Na₃VO₄, 1 mM sodium fluoride, 1 mM PMSF, and a protease inhibitor cocktail (Roche, Basel, Switzerland), and the protein concentrations in the lysates were quantified using a BCA protein assay kit (Thermo Scientific, 23227). For immunoprecipitation, 1 mg of each lysate was incubated with 2 μg of the indicated antibody or normal rabbit IgG at 4 °C overnight and then with 20 μl of Protein A/G PLUS Agarose (sc-2003, Santa Cruz Biotechnology) at 4 °C for 1 h. The agarose was washed three times with wash buffer (0.1% NP40 in phosphate-buffered saline (PBS)).
Mitochondrial isolation was performed using H1299 cells and a Mitochondrial Isolation Kit according to the manufacturer’s protocol (Thermo Scientific, 89874). The lysates were then subjected to immunoblotting using specific antibodies. Immunoblot signals were detected using an Enhanced Chemiluminescence (ECL) Kit (Millipore).

The experiment was conducted according to the method reported by Price et al. (30 (link)). Specifically, the total protein of peritoneal macrophage of BALB/c mice was extracted using radio immunoprecipitation assay lysis buffer according to the instruction of manufacture. After incubation of macrophages in 6-well plates (1 × 10⁵ cells/mL) for 36 h, the macrophages were used for the protein extraction. All the primary antibodies were diluted with PBS for 1000 times (Cell Signaling Technology, Danvers, MA, USA).
In brief, cell lysates were subjected to 10% SDS-PAGE and transferred to nitrocellulose NC membranes, and then incubated overnight at 4°C with anti-TLR4, anti-TRIF, anti-TRAF6, anti-P-NF-kB p65, and anti-GAPHD monoclonal antibodies after a 1 h blocking on (5% (w/v) non-fat milk. The membranes were subsequently washed with Tris Buffered Saline Tween (TBST) and incubated for 1 h at room temperature with corresponding secondary anti-bodies. Immunoreactive bands were detected using enhanced chemiluminescence (ECL) kit (Millipore Co., Billerica, MA, USA), GAPHD was used as internal control.

Western blotting assay was carried out as described^{45 (link),46 (link)}. Briefly, tissue or cell lysates were prepared in Laemmli Sample Buffer. Equal amounts of total proteins were subjected SDS-PAGE and transferred to PVDF membranes, which were blocked and incubated overnight with the primary antibodies against Akt (Santa Cruz Biotechnology), p-Akt (Santa Cruz Biotechnology), Col1a (Abcam), fibronectin (Abcam), α-Sma (Abcam), or Gapdh (Abcam). After being washed, the membranes were incubated with respective secondary antibodies conjugated with horseradish peroxidase (HRP). Immune-reactive signals were visualized by the Enhanced Chemiluminescence (ECL) kit (Millipore, America) on Syngene PXi6 Access imaging system (Frederick, MD). The band intensities were quantified using Image Pro Plus 6.0.

Cellular proteins were extracted, and western blotting was performed as previously reported (Mao et al., 2017 (link)). Briefly, total proteins were isolated from BMSCs with radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology, Beijing, China) containing protease inhibitors (Abcam, Cambridge, United Kingdom). Membranes were incubated at 4°C overnight with primary antibodies against RUNX2 (1:2,000; Cell Signaling Technology, Danvers, MA, United States), OCN, OPN, and SORBS1 (1:1,000; Proteintech, Wuhan, China). GAPDH (1:3,000; Cell Signaling Technology, Danvers, MA, United States) was used as the internal control. Appropriate secondary antibodies (1:3,000; Cell Signaling Technology, Danvers, MA, United States) were incubated with the blots at 20–25°C for 1 h. After rinsing with Tris-buffered saline (TBS) containing 0.05% (w/v) Tween-20 (TBST), the signals were detected using an enhanced chemiluminescence (ECL) kit (Millipore, Darmstadt, Germany) and a chemiluminescence system (Bio-Rad Laboratories, Hercules, CA, United States) and analyzed with Image Lab v6.0 software.¹

The extraction protocol for total protein quantification of fresh NP tissues was described above. After adjusting the protein concentration, 20 μg tissue lysates were separated by 12% SDS-PAGE gel using electrophoresis and transferred to PVDF membranes. The membranes were incubated with primary antibodies (p53, 1:1000, Abcam, ab26; p21, 1:1000, Abcam, ab109520; p16, 1:1000, Abcam, ab51243; p53, 1:1000, Abcam, ab26; SIRT1, 1:1000, CST, 8469s; FOXO1, 1:1000, CST, 2880s; ac-FOXO1, 1:1000, Affinity Biosciences, AF2305; GAPDH, 1:5000, CST, 5174s) overnight at 4 after blocked by 5% BSA at room temperature for 2 h. The membranes were rinsed 3 times with TBST and incubated with horseradish peroxidase (HRP)-labeled secondary antibody (1:5000, Sanggon Biotech, China) at room temperature for 2 h. The final results were acquired using the ChemiDoc MP system (Bio-Rad, USA) with enhanced chemiluminescence (ECL) kit (Millipore, USA) and were quantified using the ImagePro plus 6.0.

Western blot analysis was carried out as previously described [52 (link)]. Briefly, equal amounts of proteins (20 µg) transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA) were blocked for 1 h at room temperature and incubated overnight at 4 °C with the primary antibodies. The membrane was incubated with diluted horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000, Jackson ImmunoResearch, West Grove, PA) for 2 h at room temperature was treated with the enhanced chemiluminescence (ECL) kit (Millipore, Bedford, MA). The bound antibodies were detected using an enhanced chemiluminescence (ECL) kit and the ProteinSimple detection system (ProteinSimple Inc., Santa Clara, CA, USA).

Bovine serum albumin (BSA), 2',7’-Dichlorodihydrofluorescein diacetate (DCFH-DA), resveratrol, and DL-Glyceraldehyde were purchased from Sigma (St. Louis, MO, USA). DMEM/F12 media, fetal bovine serum (FBS), penicillin, streptomycin, and Hanks Balanced Salt Solution (HBSS) were purchased from Invitrogen (Carlsbad, CA, USA). APP 22C11 antibody was purchased from Chemicon (Temecula, CA, USA). Sirt1, NQO-1, and GRP78 antibodies were purchased from Epitomics (CA, USA). APP, Actin antibodies, and enhanced chemiluminescence (ECL) kit were purchased from Millipore (Billerica, MA, USA); and p53, Nrf-2, Ho-1, bcl-2, bax, caspase3, and β-amyloid antibodies were purchased from Santa Cruz biotechnology (Santa Cruz, CA, USA). Nitrocellulose membranes were purchased from PALL corp. (Ann Arbor, MI, USA).