Image studio ver 3.1.4

Cellular proteins were analyzed by Western blot. Briefly, after 48hr treatment, cells were collected and rinsed with ice-cold HBSS. Cells were lysed in cold RIPA lysis buffer supplemented with protease/phosphatase inhibitor cocktail (Roche, Indianapolis, IN, USA). Total protein was measured using the BCA assay (Pierce, Rockford, IL, USA). For western blots, samples were prepared by adding 2× Laemmli Sample Buffer (with 5% β-mercaptoethanol) to cell lysates. Samples were heated at ∼70°C for 10 minutes, then equal amounts of protein were loaded (20∼50ug/well) on an 8% PAGE-SDS mini-gel. Immunoblotting was performed with PVDF membranes (Bio-Rad, Hercules, CA, USA), blocked using 5% non-fat milk in TBS buffer for at least 1hr then incubated at 4°C with the respective primary antibody overnight; anti-PGP primary antibodies (Abcam, Cambridge, UK, 1:250), or anti-iNOS antibody (Cayman, Ann Arbor, MI, USA, 1:1000). Whole cellular protein was normalized using GAPDH (Cell Signaling, Danvers, MA, USA, 1:10,00) or β-Actin (Cell Signaling Danvers, MA, USA, 1:2000). The secondary antibody (IRDye 800CW goat anti-rabbit) [1:5,000] was incubated in the dark at room temperature for 45 minutes. Dual-channel infrared scan and quantitation of immunoblots were conducted using the Odyssey Sa infrared imaging system with Image Studio (Ver. 3.1.4) (LI-COR, Lincoln, NE, USA).