Mini protean 3 electrophoresis cell

Western blot analysis was performed as described
previously (29 (link)). Briefly, proteins were separated by 12%
SDS-PAGE electrophoresis at 100 V for 2 hours using
a Mini-PROTEAN 3 electrophoresis cell (Bio-Rad,
Hercules, CA, USA) then transferred to a polyvinylidene
difluoride (PVDF) membrane by wet blotting (Bio-Rad,
Hercules, CA, USA). Membranes were blocked for 1 hour
using 5% bovine serum albumin (BSA, Sigma- Aldrich,
USA), and incubated for 1.5 hours at room temperature
(RT) (30 ) with the following primary antibodies [anti-
His6 (provided with Ni-NTA Fast Start Kit (Qiagen, USA)
1:5000]. Membranes were rinsed 3 times (15 minutes
each) with Phosphate-buffered saline Tween-20 (PBST,
0.05%) and incubated with the peroxidase-conjugated
secondary antibody [anti-mouse (Millipore, 1:6000)],
for 1 hour at RT. The blots were visualized using Sigma
detection reagents (Sigma- Aldrich, USA) and films were
scanned by a densitometer (GS-800, Bio-Rad, USA).

The expression of NF-κB was analyzed by Western blotting. We separated 50 μg protein on 6.25% sodium dodecyl sulfate–polyacrylamide gels (SDS–PAGE) using the mini-PROTEAN 3 electrophoresis cell (Bio-Rad Laboratories, Inc., Hercules, California, USA) according to the manufacturer’s protocol. After being transferred to a nitrocellulose membrane, the proteins were blocked for 1 h at room temperature with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBST). The membrane was then incubated with anti- NF-κB antibody for 1 h, washed four times with TBST, and incubated for 1 h with a peroxidase-conjugated secondary antibody (1:1000). After washing the membrane four times with TBST buffer, the blot was developed using ECL chemiluminescence reagents (Millipore immobilon^TM HRP Substrate) and imaged. The immunoreactive bands on the autoradiography films were scanned with a calibrated densitometer ChemiDoc^TM XRS+ (Bio–Rad imaging System) and quantified using QuantityOne imaging software (Bio–Rad Laboratories, Hercules, CA). Equal loading of proteins onto the gel was confirmed by the immunodetection of GAPDH.

Nuclear erythroid factor-2 (Nrf-2), NF-κB, and pNF-κB total and phosphorylated (NF-κB and pNF-κB, respectively), collagen I, collagen III, and caspase-3 expression were determined. LV samples were extracted using radioimmunoprecipitation assay buffer or nuclear extraction buffer. Protein content was quantified using the Bradford method [43 (link)], and samples containing 50 μg of protein were separated by electrophoresis using a Mini-Protean 3 Electrophoresis Cell (Bio-Rad, Hercules, CA, USA) system and transferred to a nitrocellulose membrane. The membrane was blocked and incubated with primary antibodies. The membrane was then washed with TBS and Tween 20 and incubated with the appropriate secondary peroxidase-conjugated antibody. A SuperSignal® West Pico Chemiluminescent Substrate (Pierce Protein Research Products, Rockford, IL, USA) was used to detect bound antibodies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal (Santa Cruz Biotechnology Inc., Europe) was used for normalization.