Ab201015

Proteins from 16HBE cells were obtained and bicinchoninic acid assay was performed to detect the protein concentration. After that, equal quantity of proteins (50 µg) were solubilized in 5× sodium dodecyl sulfate (SDS)-sample buffer and separated on the SDS polyacrylamide gels (Thermo Fisher Scientific, Inc.). Separated proteins were then transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). After blocking, the membranes were incubated with anti-IDO (dilution, 1:500; ab55305); anti-p-Raf-1 (dilution, 1:500; ab208449); anti-Raf-1 (dilution, 1:1,000; ab50858); anti-p-Mek1/2 (dilution, 1:1,000; ab194754); anti-Mek1/2 (dilution, 1:1,000; ab215263); anti-p-Erk1/2 (dilution, 1:1,000; ab201015); anti-Erk1/2 (dilution, 1:1,000; ab17942); anti-GAPDH (dilution, 1:1,000, ab8245; all from Abcam) antibodies overnight at 4°C. After that, horseradish peroxidase-conjugated secondary antibodies (bs-0293M; BIOSS, Beijing, China) were added and incubated at room temperature for 1 h. The results of all the assessments were evaluated by enhanced chemiluminescent reagents (EMD Millipore).

Hippocampal neurons were washed twice with ice-cold phosphate-buffered saline and then lysed with ice-cold lysis buffer (20 mM Tris-HCl (pH=7.5), 150 mM NaCl, 1 mM Na3VO4, 1 mM PMSF, 1 mM EDTA, 1% NP40, 50 mM NaF) for 30 min. Cell lysates were centrifuged at 10,000 x g for 15 min at 4°C. Cell lysates were separated by sodium dodecyl sulfate-polyacrylamide 12% gel electrophoresis and transferred to polyvinylidene fluoride membranes. Following incubation with blocking buffer (Tris-buffered saline + 0.1% Tween-20 (TBST) + 5% non-fat milk), membranes were probed with rabbit anti-Nrf2 polyclonal antibody, (1:1,000, ab31163 abcam, Cambridge, UK) mouse anti-ERK1+ERK2 polyclonal antibody (1:1,000, ab54230, abcam), rabbit anti-ERK1 (phospho T202) + ERK2 (phospho T185) antibody (1:1,000, ab201015, abcam) and mouse anti-GAPDH monoclonal antibody (1:2,000, ab8245, abcam) overnight at 4°C. The membranes were then washed with TBST and incubated with a horseradish peroxide-conjugated secondary antibody (1:1,000, 00001–2, ProteinTech Group, Inc., Chicago, IL, USA), and signals were detected with an enhanced chemiluminescent reagent (EMD Millipore, Billerica, MA, USA). Images were captured using a Bio-Rad VersaDoc 3000 (Bio-Rad Laboratories, Inc., Gladesville, NSW, Australia) and protein levels were quantified using Image-pro Plus 6.0 (Media Cybernetics, MD Rockville, USA).