Halt proteinase phosphatase inhibitors

EBV-B cells were treated for 3 hours with PapMV nanoparticles or rapamycin, with or without pretreatment with 5 mM 3-MA. Cells were collected and washed Dulbecco’s phosphate-buffered saline (D-PBS, Wisent Bioproducts). Proteins were extracted in the presence of HALT proteinase/phosphatase inhibitors (ThermoFisher) from the above-mentioned pelleted cells and quantified by Bio-Rad protein assay. Proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad). Membranes were incubated with rabbit an anti-human LC3B antibody (Ab; 1:3,000) (Novus Biologicals), a rabbit anti-ATG5-specific Ab (Cell Signaling) or a mouse anti-human β-actin-specific Ab (1:10,000) (Millipore). Membranes were revealed with horseradish peroxidase (HRP)-linked anti-rabbit (1:5,000) or anti-mouse (1:40,000 for β-actin) Abs (Chemicon) followed by enhanced chemiluminescence (ECL) prime detection (Amersham) on film. Densitometry was performed with ImageJ software and is reported as the ratio between densities of LC3-II over LC3-I bands or the ratio of ATG5 over β-actin bands.

Proteins were prepared in the presence of HALT proteinase/phosphatase inhibitors (Thermofisher) from the above-mentioned pelleted cells, quantified, resolved on 10% SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad). The membranes were incubated with anti-human IDO antibody (Ab) (Hycult Biotechnology) or anti-human β-actin-specific mouse monoclonal Ab (Chemicon) [4] (link). For total STAT1, and Y701 or S727 phosphorylated forms, proteins were resolved on 7.5% SDS-PAGE, incubated overnight at 4°C with rabbit polyclonal Ab (anti-STAT1, anti-Y701 and anti-S727 phospho-STAT1, all from Cell Signaling). IDO antibody specificity was confirmed using a plasmid encoding IDO which was transfected into MDA-231 cells using lipofectamine and plus reagent following manufacturer' instructions (Life technologies; Figure S1).