Amersham ecl plus kit

Cells grown in 6-well dishes were treated with OA and/or HTyr as indicated above and lysed as previously described [12 (link)]. The extracts were boiled for 5 min and samples containing an equal amount of total protein (25 μg) were loaded on 10% SDS-polyacrylamide gels. Following electrophoresis, the proteins were transferred onto a nitrocellulose membrane [13 (link)]. To detect ACC, FAS, and HMGCR, membranes were incubated with the specific primary antibodies for 1.5 h at room temperature and then for 1 h with appropriate horseradish peroxidase-conjugated IgG (dilution 1 : 5000). Signals were detected by enhanced chemiluminescence using the Amersham ECL plus kit (GE Healthcare, Milan, Italy). Beta-actin detection was used for signal normalization.

Extracellular ROS production was determined by measuring ROS released into the medium using an Acridan Lumigen PS-3 assay 39 (link). RAW 264.7 cells (5 × 10³ cells/well) were seeded in clear-bottom, 96-well black plates and treated with different concentrations of LPS (0, 1, 5, 10 μg/ml) for 1 hour. Thereafter, 50 μl aliquots of medium were mixed with the same volume of a 40:1 ratio of Reagent A (H2O2 in Tris buffer) and Reagent B (acridan solution in dioxane and ethanol) in an Amersham ECL Plus kit (GE Healthcare), and then incubated for 5 minutes at RT with light protection. Luminescence intensity at an emission wavelength of 430 nm was measured using a microplate reader (SpectraMax Gemini XPS; Molecular Devices). Intracellular ROS were measured using 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) dye, according to the manufacturer's instructions. After incubating for 24 hours in clear-bottom, 96-well black plates, RAW 264.7 cells were incubated with 20 μM DCFDA dye for 45 minutes at 3 °C in the dark and then washed gently with medium. Thereafter, PBS or 5 μg/ml LPS, with or without each SPION (Fe, 1 mM), was added, and fluorescence intensity was measured at excitation and emission wavelengths of 485 and 535 nm, respectively, using a fluorescence microplate reader (SpectraMax Gemini XPS; Molecular Devices).

The detailed Western blot method has been previously described [30 (link)]. Briefly, the front part of brain protein extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The antibodies used were as follows: anti-claudin-5 (ab53765) (1:500; Abcam), GAPDH (sc-32233) (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-gp91phox (sc-20782) (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were incubated for 1 h with appropriate secondary antibodies and processed with an enhanced chemiluminescence reagent kit (Amersham ECL Plus kit, GE Healthcare, UK). Mouse or rabbit secondary antibody (1:5000 or 1:30000; Cell Signaling) was used in accordance with first antibody. The antibodies were visualized by using an enhanced chemiluminescence method (ECL Plus; GE Healthcare, Buckinghamshire, UK). The intensity of the bands was quantified by using analysis software (Image J; National Institute of Health, Bethesda, MD, USA). In individual samples, each value was corrected for that of GAPDH.

Equal amounts of proteins (20 μg), as determined by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA), were resolved on a 8% or on a 12% SDS-PAGE and electrotransferred to Amersham Hybond PVDF membrane (GE Healthcare, Milan, Italy). Membranes were then blocked for 1 h at 25 °C in PBS containing 5% nonfat dry milk and 0.1% Tween-20 (Sigma-Aldrich), and were incubated for 1 h at 25 °C or overnight at 4 °C, with the following primary antibodies: BRLF1 and BZLF1(1 : 200 and 1 : 100, respectively, both obtained from Argene Biosoft, Verniolle, France), BALF5 (1 : 100), LC3 (1 : 8000, L7543) and β-actin (1 : 5000) were purchased from Sigma, Beclin1(1 : 500; sc-10086) and ATG5 (1 : 200, sc-133158) purchased from Santa Cruz (DBA, Milan, Italy), and phospho-p70 S6 kinase (1 : 1000, 07-018-I Millipore, Merk Spa, Vimodrone, MI, Italy). Phospho ERK1/2 and ERK1/2 antibodies (1 : 500) were obtained from Cell Signaling (EuroClone, Milan, Italy). The membranes were then incubated with the appropriate secondary antibodies conjugated to HRP (1 : 7500, Bio-Rad). The specific signals, visualized by Amersham ECL Plus kit (GE Healthcare) were quantified by densitometric analysis by ImageJ free-share software (http://imagej.nih.gov/ij).

Proteins were separated by 10% SDS/PAGE and transferred onto a nitrocellulose membrane. Blocking and incubation with primary and secondary antibodies were performed in 5% (w/v) nonfat dry milk. Primary antibody against phospho‐Darpp‐32/t‐Darpp (T39) (#2301) was from Cell Signaling Technology (Danvers, MA, USA). H62 (sc‐11365), an antibody that recognizes both Darpp‐32 and t‐Darpp, was from Santa Cruz Biotechnology (Dallas, TX, USA). Horseradish peroxidase‐conjugated anti‐rabbit IgG secondary antibody was purchased from Cell Signaling Technology. Chemiluminescence was produced using the Amersham ECL Plus kit (GE Healthcare Bio‐Sciences, Pittsburgh, PA, USA) and captured on X‐ray film.

Cells were lysed by treating with RIPA lysis buffer (Life Technologies, USA). Cell lysates were run in precast SDS-PAGE 4–15% gels (Novagen, Madison, WI, USA) and blotted on nitrocellulose membranes (Novagen). Membranes were washed with Tris-buffered saline (TBS) and blocked with fat-free milk in the same buffer for 1 hour at room temperature. The membranes were treated overnight at 4°C with primary antibodies directed against proteins or phospho-proteins of interest at dilutions as recommended by the manufacturers as listed in Supplementary Table 3. The next day, the membranes were washed and incubated with horseradish peroxidase (HRP) conjugated secondary antibodies at the recommended dilutions as described in Supplementary Table 3. Finally, the membranes were developed by using the Amersham ECL Plus kit (GE Healthcare, Piscataway, NJ, USA) per the manufacturer’s protocol.

[LOOSESTCells were lysed using NP40 cell lysis buffer (Life Technologies). Cell lysates were separated by SDS-PAGE using a precast 4–15% gel (Bio-Rad, Hercules, CA) and blotted on nitrocellulose membranes (Bio-Rad, Hercules, CA) that were then washed with Tris-buffered saline (TBS) and blocked with fat-free milk in the same buffer for 1 hour at room temperature. The membranes were subject to incubation with a primary rat monoclonal antibody directed against mouse CCR6 (clone CKR-6 (13Q7), dilution 1∶200) or a mouse anti-β-actin antibody (clone ACTBD11B7, dilution 1∶1000) (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4°C. The following day, the membranes were washed and incubated with goat anti-rat IgG-HRP conjugated secondary antibody (for anti-CCR6 primary antibody) and secondary goat anti-mouse IgG-HRP (for anti-β-actin primary antibody) (both at 1∶5000 dilution, Santa Cruz Biotechnology, Inc.). Finally, the membranes were developed by using the Amersham ECL Plus kit (GE Healthcare, Piscataway, NJ) as per the manufacturer's protocol.