Ab50858

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).