Odyssey imaging software v3

Platelets treated with TRAIL or PBS were washed and homogenized on ice using RIPA buffer (50 mM TRIS pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 1 mM phenylmethanesulfonyl fluoride). Lysates containing equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membranes using a semi-dry transfer system (Bio-Rad, Hercules, CA, USA). Blots were blocked with 5% nonfat dry milk in TBST (10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) for 2 hours at room temperature, and then probed with mouse monoclonal antibody against DR5 (1.5 µg/mL, ab16329, Abcam, Cambridge, MA, USA), mouse monoclonal antibody against DR4 (1.5 µg/mL, ab8414, Abcam), and rabbit polyclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:800; sc-32233; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. The blots were then washed with TBST and incubated with fluorescence-conjugated goat anti-mouse/rabbit IgG (Rockland Immunochemicals Inc., Gilbertsville, PA, USA) for 1 hour at room temperature. The immunoreactive bands were visualized using an Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA), and protein expression was quantified by densitometric analysis using Odyssey Imaging Software v3.0 (Li-Cor Biosciences).