Antibodies against β actin

Tumour slices treated as indicated were kept at −20 °C for immunoblotting. Total extracts were prepared as described previously and loaded on polyacrylamide gels (SDS-PAGE) and transferred to nitrocellulose membranes [11 (link)]. Antibodies against Extracellular Regulated Kinase 1/2 (ERK) and phosphorylated ERK1/2 (pERK) were from Cell Signaling Technology (Danver, MA, USA). Antibodies against β actin were from Sigma (Saint-Quentin Fallavier France). Secondary antibodies coupled to peroxydase were from GE Healthcare (Aulnay-sous-Bois, France). Enhanced chemiluminescence reaction was used for revelation. Immunoblots were scanned and quantified using the software Image J (National Institute of Health, USA).

PA-2 was provided by Medicon Pharmaceuticals, Inc., Setauket, NY. Aspirin were purchased from Sigma (St Louis, MO). For cell culture study, we prepared 500 mM stock solutions of both in DMSO. In all cell culture media, the final DMSO concentration was adjusted to 1%. All general solvents and reagents were of HPLC grade or of the highest grade commercially available. Antibodies against β-actin were from Sigma. All other antibodies were from Cell Signaling (Beverly, MA).

For protein detection, 25,000 cells/cm² were seeded in a 6-well plate and exposed to NPs at the indicated concentrations for 24 h. Following exposure, cells were collected and lysed overnight at 4°C in RIPA buffer [50 mM Tris HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 0.1% SDS, 1 mM EDTA]. Protease- and phosphatase inhibitors (Mini EDTA-free Protease Inhibitor Cocktail, Sigma Aldrich; 1 mM PMSF, Thermo Fisher; PhosSTOP™, Sigma Aldrich) and 1 mM DTT (Sigma Aldrich) were freshly added to the buffer. Cell lysates were centrifuged at 13.000 × g for 15 min and supernatants were collected. The protein concentration was measured using the Bradford assay and 30 µg were loaded into each well of a NuPAGE 4%–12% Bis-Tris gradient gel (Thermo Fisher). Following electrophoretic separation, the proteins were transferred to a Hybond Low-fluorescent 0.2 µm PVDF membrane (Amersham), blocked for 1 h in Odyssey^® Blocking Buffer (PBS) (LI-COR), and stained overnight at 4°C with primary antibodies against NRF2 (Abcam, ab62352). Antibodies against β-actin (Sigma-Aldrich) were used for loading control, and the goat anti-mouse IRDye 680RD antibody (LI-COR Biotechnology GmbH, Bad Homburg, Germany) was used as a secondary antibody. Proteins were detected using LI-COR Odyssey^® CLx scanner and Odyssey^® Image Studio software.

Kidney and liver tissues were homogenized in lysis buffer containing 300 mM NaCl, 50 mM Tris–HCl, 0.5% Triton X-100, and protease-inhibitor cocktail (pH 7.6). The incubation was performed at 4 °C for 30 min. After centrifugation at 14,000 rpm for 20 min at 4 °C, protein concentrations of the lysates were determined with the Bradford protein assay reagent (Bio-Rad, Hercules, CA). The proteins (40 μg) were loaded into 7.5–15% SDS/PAGE gels and transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The membranes were incubated overnight in blocking buffer at 4 °C with each antibody. Antibodies against sterol regulatory element-binding protein (SREBP)-1 and liver X receptor (LXR) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against SCD-1 and Elovl-6 were purchased from Abcam (Cambridge, MA). Antibodies against β-actin were obtained from Sigma (St. Louis, MO). The membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibody for 60 min at room temperature. Immunostaining with antibodies was performed using the Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Hudson, NH) and detected with the LAS-3000 Plus (Fuji Photo Film, Tokyo, Japan). The samples were quantified and normalized against the β-actin control using ImageJ version 1.48.

GTS-21, α-bungarotoxin, and antibodies against Bcl2 and PGC-1α were purchased from Abcam (Cambridge, UK). LPS (Escherichia coli serotype 055:B5), methyllycaconitine, and antibodies against β-actin and BDNF were purchased from Sigma-Aldrich (St. Louis, MO, USA). MPTP was obtained from Tokyo Chemical Industry Co. (Tokyo, Japan). Antibodies against Iba-1 were purchased from Wako (Osaka, Japan). Antibodies against phospho-/total forms of AMPK, Akt, MAP kinases, CREB, and antibodies for IL-6, TGF-β, NQO1, PPAR-γ, and TH were obtained from Cell Signaling Technology (Beverley, CA, USA). While antibodies against phospho-p47phox were provided by Assaybiotech (Sunnyvale, CA, USA), those against TNF-α, Nrf2, HO-1, catalase, lamin A, and COX-2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against iNOS and IL-1β were purchased from BD Biosciences (San Jose, CA, USA), and an antibody for 4-hydroxy-2E-nonenal (HNE) was purchased from Alpha Diagnostic International (San Antonio, TX, USA).

Total protein lysate (20 μg) was separated on a 10% SDS-polyacrylamide gel and then transferred to Immobilon–P membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat skim milk before being incubated with primary antibodies at 4 °C overnight. Antibodies against β-actin were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Anti-CAR and anti-CD46 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). After being incubated for 1 h with secondary antibodies, proteins were detected and analyzed by Immobilon (Millipore, Billerica, MA, USA) and ChemiDOC ^TM MP Imaging System (Bio-Rad, Hercules, CA, USA).