Hybond p pvdf

RIPA buffer (Thermo Fisher Scientific, Inc., Rockford, IL, USA) was used for preparation of whole cell extracts and the protein concentration was measured by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of cell lysates were separated on SDS-PAGE and then transferred onto Hybond-P PVDF (GE Healthcare, Buckinghamshire, UK) using a CAPS transfer buffer at 30 mA overnight at 4 °C. The membranes were blocked in a freshly made blocking buffer (5% skim milk in PBS with 0.05% Tween 20, pH 7.4, PBS-T) for 6 h at room temperature. After washing with PBS-T, the membranes were incubated with an appropriate dilution (1:1000‒1:5000) of primary antibody (Table 1) overnight at 4 °C on a rocking platform. The membranes were then washed and incubated with suitable horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA, at a dilution of 1:10,000‒1:25,000) for 1 h at room temperature. The blots of were incubated by ECL Prime (GE Healthcare), and the chemiluminescent signals were then visualized with X-ray film. Densitometry of the bands was analyzed by ImageJ software version 1.50 (National Institutes of Health, Bethesda, MD, USA).

Enzymatic protein levels were evaluated as previously reported [47 (link)] by resolving 35 µg of each protein sample by SDS-PAGE (12%). The gels were electroblotted onto hydrophobic polyvinylidene difluoride membranes (Hybond-P PVDF, GE Healthcare Bio-Science, Uppsala, Sweden). Antibody staining was performed with a chemiluminescence detection system (ECL Plus Western Blotting Detection Reagent, GE Healthcare Bio-Science, Uppsala, Sweden) using a 1:500 dilution of the mouse anti-human TS (TS106) monoclonal primary antibody (Invitrogen S.r.L., Milan, Italy), 1:1000 dilutions of the mouse anti-human DHFR monoclonal antibody (Tebu-Bio, Milan, Italy), and 1:1000 of mouse anti-human ß-actin antibody (Sigma-Aldrich S.r.L., Milan, Italy) in conjunction with a 1:3000 dilution of horseradish peroxidase-conjugated sheep anti-mouse secondary antibody (GE Healthcare Bio-Science, Uppsala, Sweden). Quantification of signal intensity was performed by densitometry on a GS-800 calibrated densitometer (Bio-Rad) and analysed by using Quantity One software (Bio-Rad, Hercules, CA, USA).

Confluent RAW 264.7 cells (1 × 10⁶/mL) were incubated with vehicle, LPS plus vehicle, or LPS plus test sample (0.05 and 0.1 mg/mL). After being incubated for 16 h, cell lysate was prepared using RIPA buffer (Thermo Fisher Scientific, Inc., Rockford, IL, USA) and the protein concentration was determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA) using bovine serum albumin as a standard.
Equal amounts of cell lysates were separated on SDS-PAGE (8%–12%) and then transferred onto Hybond-P PVDF (GE Healthcare, Buckinghamshire, UK) using a CAPS transfer buffer at 20 volt overnight at 4 °C. The membranes were blocked at room temperature in a freshly made blocking buffer (5% skim milk in PBS with 0.05% Tween 20, pH 7.4) for 6 h. The membranes were then incubated overnight at 4 °C in blocking buffer containing appropriate dilution (1:1000–1:5000) of primary antibody (Table 3). The membranes were then incubated for 1 h at room temperature with suitable horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) at a dilution of 1:10,000–1:25,000. The proteins of interest were detected by ECL Prime (GE Healthcare) and the chemiluminescent signals were then visualized with X-ray film. Densitometry of the bands was analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA).

PC12 cells (1×10⁶/ml) were seeded on 100 mm dishes in serum-free medium and exposed to the indicated reagent for the indicated period. Cells were washed with PBS, scraped with ice cold RIPA buffer (Thermo Fisher Scientific, Inc., Rockford, IL) and then incubated on ice for 30 min. The cellular debris was removed by centrifugation (8,000×g for 15 min) at 4°C and the cell lysate was carefully transferred to the microcentrifuge tube. The protein concentration was measured by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA) using bovine serum albumin as a standard.
Cell lysates were separated on 10% SDS-PAGE and transferred onto Hybond-P PVDF (GE Healthcare) at 20 volts overnight at 4°C. The membranes were blocked at 4°C in PBST blocking buffer (5% BSA in PBS with 0.05% Tween 20, pH 7.4) for 8 h. Blots were analyzed with each antibody (Table 2) at a dilution of 1∶1000–1∶5000 overnight at 4°C. After three washes with PBST, the blots were incubated with suitable horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) at a dilution of 1∶10,000 for 1 h. The blots were washed again and the proteins of interest detected by Amersham ECL Prime Western Blotting Detection Reagents (GE Healthcare), according to the manufacturer’s instructions, and then the chemiluminescence signal was visualized with Hyperfilm ECL X-ray film.