Gel pro analyzer 3

NCM460 and HepG2 cell lines were treated with IL-6 and/or aspirin (0.5 or 1.5 mM) for 24 hours. Total RNA was extracted starting from the same cell number. The 28S and 18S RNA subunits were visualized by loading RNA in a 1% agarose gel stained with ethidium bromide. The intensity of bands was evaluated with the densitometric software GelPro analyzer 3.0 (Media Cybernetics, Silver Spring, MD).

Cells were lysed in a buffer containing 10 mM Tris-HCl pH 7.4, 150 mM NaCl, 50 mM NaF, 1% NP-40, 0.5% SDS containing protease inhibitors cocktail (Roche), 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol. Thirty micrograms of total protein were separated by SDS-PAGE and electro-blotted onto nitrocellulose (0.45μm, Bio-Rad) or PVDF (0.2 μm, EMD Millipore) membrane. The membranes were blocked in 5% skimmed milk (Santa-Cruz) and probed with primary antibodies. The blots were washed, incubated with secondary horseradish peroxidase-conjugated secondary antibodies and developed by enhanced chemiluminescence (ECL, ThermoFisher Scientific). Western blot densitometry was performed using Gel-Pro Analyzer 3.0 software (Media Cybernetics). The optical densities were normalized to those of control samples.

The protein expression of GDNF and NGF was investigated by western blot analysis. The specimen for each groups were placed into a 24-well plate and then Schwann cells were seeded on the surface of disks at the density of 2 × 10⁵ cells per well. After incubation for 48 h, the protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). Equal amounts of protein (40 μg) were resolved by SDS-PAGE electrophoresis, and then transferred to polyvinylidene fluoride film (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk in TBST (0.1% Triton X-100 in TBS) buffer for 90 min, and then incubated with primary antibodies against NGF (rabbit anti-rat, 1:1000, cat. no. WL0151, Wanleibio, Shenyang, China) and GDNF (rabbit anti-rat, 1:400, cat. no. PB0045, Boster Biological Technology, Ltd, Wuhan, China) overnight at 4 °C. Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit, 1:5000, cat. no. A0208, Biyuntian, China) and visualized with an ECL reagent (Qihai Biotech, China). The intensity of the bands, which were representative of protein levels, were assessed using Gel-Pro-Analyzer 3.0 (Media Cybernetics, Rockville, MD, USA).β-actin was used to normalize target proteins [33 (link), 34 (link)].