Ecl western blotting system

Kidneys were lysed in solubilization buffer containing 20 mM HEPES (pH 7.2), 1% Triton X-100, 10% glycerol, 20 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM PMSF, 10 μg/mL aprotinin, and 10 μg/mL leupeptin. Insoluble material was removed by centrifugation (10,500 g, 10 min). Lysates (60 μg) were resolved by SDS-PAGE and transferred to PVDF membranes (Immobilon, Millipore, Bedford, MA). Nonspecific binding sites were blocked in TBS buffer (10 mM Tris・HCl, pH 7.4, 0.15 M NaCl) containing 0.1% Tween 20 and 5% BSA overnight at 4ºC or for 1 h at room temperature. Antibodies were added to TBS containing 0.1% Tween 20 with 5% BSA and incubated with mixing for 24 h at 4°C or for 2 h at room temperature. After incubating with secondary antibody, bound antibodies were detected using the ECL Western blotting system (Amersham, Arlington Heights, IL). All experiments were repeated at least three times on separate samples.

Proteins were extracted from the cytoplasm and nucleus separately using NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s protocol. The extracted nucleus protein (20 μg) and cytoplasmic protein (20 μg) with sample buffer containing 2-mercaptoethanol was separated on 12.5% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA) and then incubated with 5% skim milk overnight. The membrane was then incubated overnight with primary antibodies against NF-κB p65 (Cell Signaling Technology, Beverly, MA, USA), Lamin A⁄C (Santa Cruz Biotechnology, Dallas, TX, USA), beta-actin (Sigma-Aldrich, St. Louis, MO, USA), Bcl-2 (Santa Cruz Biotechnology, Dallas, TX, USA) and survivin (Santa Cruz Biotechnology, Dallas, TX, USA). After incubation with appropriate secondary antibodies, signals were visualized using an ECL Western blotting system (Amersham, Piscataway, NJ, USA).

Twenty micrograms of proteins were separated by SDS-PAGE. Low-range molecular weight standards were used (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were then transferred to a nitrocellulose membrane by electroblotting and incubated with a mouse monoclonal anti-GFP antibody (Roche Diagnostics AG, Basel, Switzerland). Membrane was then incubated with the corresponding secondary antibody coupled to horseradish peroxidase (Promega, Madison, WI, USA) and developed by enhanced chemiluminiscent staining using ECL western blotting system (Amersham Biosciences, Piscataway, NJ, USA).

For the extraction of total cellular protein, cells were washed with ice-cold PBS and lysed with lysis buffer (50 mM Tris–HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA [pH 7.4], 1% NP-40 with protease and phosphatase inhibitors cocktail) treated for 5 min at 96 °C and centrifuged at 20,000 × g for 15 min. Protein concentrations were estimated by Pierce BCA protein assay (Thermo Fisher Scientific). The whole-cell lysate was mixed with a 1/5 volume of 5 × SDS sample buffer and boiled. Total cell proteins were resolved by SDS-PAGE followed by electro-transfer of proteins onto a PVDF membrane. The membrane was blocked for 1 h at room temperature (RT) using BSA/Tris buffered saline with 0.05% Tween 20 (TBST). The membrane was then incubated with primary antibodies overnight at 4 °C. After washing with TBST 3 times for 5 min, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG at 1:5000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at RT. After washing with TBST 3 times for 5 min, blots were developed using the enhanced chemiluminescence ECL Western Blotting System (Amersham).

Nuclear lysate from human PBMC was prepared as previously described by [52 (link)]. Twenty μg of nuclear proteins, quantified with the Bradford method (Bio-Rad Laboratories Inc., Hercules, CA, USA), were separated by gel electrophoresis on 4–12% Bis-Tris Criterion XT precast gels (Bio-Rad Laboratories Inc., Hercules, CA, USA) and electroblotted onto polyvinylidene fluoride membranes (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). Immunoblotting was performed with rabbit HIF-1α antibody (1:1000), anti-NRF2 polyclonal antibody raised against a peptide mapping at the C-terminus (C-20) (1:1000), rabbit anti-NF-κB (p65) polyclonal antibody (1:1000) (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-Lamin B monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA) (1:1000), followed by peroxidase-conjugated secondary antibody HRP labeled goat anti-rabbit Ig (BD Pharmigen, San Diego, CA, USA) (1:5000), goat anti-mouse IgM secondary antibody HRP conjugate (Thermo Scientific, Waltham, MA, USA) (1:10,000), and visualized with an Electrochemiluminescence (ECL)western blotting system (AmershamBiosciences, Buckinghamshire, UK).

Cells were lysed in solubilization buffer containing 20 mmol/L HEPES (pH 7.2), 1% Triton X‐100, 10% glycerol, 20 mmol/L sodium fluoride, 1 mmol/L sodium orthovanadate, 1 mmol/L phenylmethanesulfonyl fluoride, 10 μg/mL aprotinin, and 10 μg/mL leupeptin. Insoluble material was removed by centrifugation (10,500g, 10 min). Lysates were resolved by SDS‐PAGE and transferred to PVDF membranes (Immobilon, Millipore, Bedford, MA). Nonspecific binding sites were blocked in Tris‐buffered saline (TBS) (10 mmol/L Tris・HCl, pH 7.4, 0.15 mol/L NaCl) containing 0.1% Tween 20 and 5% skim milk or BSA overnight at 4°C or for 1 h at 25°C. Antibodies were added to TBS containing 0.1% Tween 20 with 5% BSA and incubated with mixing for 24 h at 4°C. The following dilution of antibodies were used: P‐p38 at 1:2000, P‐ERK at 1:1000, p38 at 1:5000, ERK at 1:1000, CDH11 at 1:125. Bound antibodies were detected using the ECL western blotting system (Amersham, Arlington Heights, IL). All experiments were repeated on at least three separate occasions. Bands were quantitatively analyzed by ImageJ software.