Detection of Phosphorylated IDO1 (pIDO1) by Western Blotting

Lung extracts from susceptible and resistant mice infected with P. brasiliensis, treated or not with 1MT were obtained after 96 h and 2 weeks of infection and analyzed by Western Blot for detection of pIDO1. Protein concentration was determined using a BCA assay (Pierce), and 20 µg of protein in Ripa Buffer and Sample buffer 4× were heated at 100°C for 4 min, then on ice for 2 min and loaded 60 μL per well in 10% SDS PAGE, 1.5 mm. Electrophoresis was then performed by transferring the gel to the nitrocellulose membrane with the membranes incubated in blocking solution [5% non-fat dry milk, 0.1% Tween 20 in Tris buffer saline (TBS) for 1 h at room temperature on an orbital shaker]. After two washings steps with TBS-Tween 20 0.1% buffer, the membrane was incubated overnight with polyclonal rabbit antimouse pIDO (CV223 AP8), 2 μg/mL in 5% non-fat dry milk, TBS at 0.1%. The membrane was washed three times for 5 min and incubated with HRP-labeled secondary antibody (anti-rabbit HRP–Pierce) for 1 h at room temperature. The revelation was performed using the ECL chemiluminescent substrate for enzymatic activity detection of peroxidase (HRP) on photographic film. The same method was used with a monoclonal rabbit anti-mouse IDO1 (CV152) and monoclonal antimouse β-tubulin antibody (1:1,000).