Gel pro analyzer software version 3

The liver tissues were dissolved by radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China), total protein was isolated and the protein concentration was determined using a bicinchoninic acid Method-Protein Quantitative Reagent kit (Beyotime). After that, tan equal quantity of protein was fractionated using 8, 10 or 13% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Bedford, MA, USA). Then, the membranes were blocked by 5% (w/v) fat-free milk and immunoblotted with primary antibodies (dilution and suppliers were listed in Table I) 4°C overnight. After washing, the membranes was incubated with HRP-conjugated secondary antibodies (goat anti-rabbit IgG, 1:5,000; goat anti-mouse IgG, 1:5,000; Beyotime Institute of Biotechnology) at 37°C for 45 min. Next, the specific protein bands was visualized using enhanced chemiluminescence reagent (Qihai Biotec, Shanghai, China) and the gray density was analyzed using Gel-Pro Analyzer software version 3.0 (Media Cybernetics, Inc., Silver Spring, MD, USA).

Total cellular RNA of lung tissues was isolated using the RNAprep Pure kit (For Tissue) (Tiangen Biotech Co., Ltd.) according to the manufacturer's protocol. Total RNA (2.0 µg) was reverse transcribed into complementary DNA using the RevertAid First Strand cDNA Synthesis kit with oligo(dT). All primers were designed and synthesized by Takara Bio, Inc. The primer sequences, annealing temperatures and expected product sizes are listed in Table I. All PCR procedures were conducted in the MJ Research PTC-200 DNA Engine Thermal Cycler (Bio-Rad Laboratories, Inc.) using the following cycling parameters: 2 min of initial denaturation at 94°C, followed by 30 sec of denaturation at 94°C, and 35 cycles of 30 sec at 94°C (denaturing), 30 sec at 58°C (annealing) and 30 sec at 72°C (elongation), with a final extension at 72°C for 5 min. The presence of PCR products was confirmed by gel electrophoresis on a 2% agarose gel and staining with ethidium bromide. Bands were visualized in a Gel Doc 1000 system (Bio-Rad Laboratories, Inc.). β-actin was used as an internal control in parallel for each replicate. Gel-Pro Analyzer software version 3.0 (Media Cybernetics, Inc., Rockville, MD, USA) was used to quantify the denisty of each band. The experiments were performed three times independently, and the mean value was used for comparison.