Rabbit anti mouse inos polyclonal antibody

To examine arginase I or iNOS expression, PC were removed from BALB/c or PD-L2 KO, TE or PBS injected mice. Additionally PC were isolated from BALB/c or PD-L2 KO infected mice. Cells were washed and lysed for 30 min at 4°C in RIPA buffer [1% Triton X-100 (v/v), 0.5% sodium deoxicolate (p/v), 0.1% sodium dodecyl sulfate (SDS)] containing protease inhibitor cocktail (Roche) and cell debris was spun down at 13,000 g for 15 min. Precipitates were removed, and aliquots of the cell lysates were diluted in SDS sample buffer, boiled at 100°C for 3 min, spun down, and applied to precast 10% acrylamide Tris-glycine gels at 40 μg protein/lane and run at 150 V for 1 h. Samples were transferred to nitrocellulose membrane (BioRad) at 100 V for 1 h and membranes were probed using rabbit anti-mouse arginase I polyclonal antibody or rabbit anti-mouse iNOS polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) followed by peroxidase conjugated anti-rabbit antibody (Sigma Chemical Co.) and visualized using enhanced chemiluminescence (Pierce) for detection. Moreover rabbit anti-mouse ATP citrate or monoclonal anti-mouse β-actin antibodies (Cell Signalling Technology) were used for control loading.

Western blot analyses were performed using the following antibodies: rabbit anti-mouse iNOS polyclonal antibody from Santa Cruz (Dallas, TX, USA) and mouse anti-Actin monoclonal antibody from Millipore Corp. (Billerica, MA, USA), and all rabbit monoclonal antibodies raised against Beclin-1, Atg5, Atg12, Atg16L, LC3A/B, and phospho-IRF-3 from Cell Signaling Tech. (Danvers, MA, USA).