PDF《differentiation》

1 g/L SDS. Membranes were blocked for one hour in

1 * TBS/0.1% NP40 with 10% newborn calf serum and 5% dry milk. The following primary antibodies were incubated with the appropriate membranes at a dilution of 1:500 overnight: anti-Bid antibody (Santa Cruz), anti-AU1 antibody (Covance), anti-FLAG (M5, Sigma), anti- TRAIL (H-257, Santa Cruz), anti-FLIP (Dave-2, Alexis) and anti-DR5 (TRAIL-R2, R&

D Systems). The actin primary antibody (Sigma) was used at a dilution of 1:30,000 for one hour. Appropriate HRP-conjugated secondary antibod- ies, each diluted 1:1000, were incubated with the membranes for one hour. After each incubation with antibody and prior to the addition of chemiluminescent substrate, membranes were washed ?ve times in

1 * TBS (tris-buffered saline pH 7.4) with 1% NP-40. Membranes were then incubated with SuperSignal West Pico Chemiluminescent Substrate (Pierce) for

60 s and bands were visualized using either Kodak Scienti?c Imaging Film or a Bio-Rad Fluor-S Multi- imager. Cytosolic nucleosome ELISA Myoblasts were plated at equal density and the next day switched to fresh GM or DM for eight hours. This is the op- timal time point for this assay as DNA fragmentation is max- imal yet apoptotic myoblasts have not yet detached from the plate [7]. Cytosolic nucleosomes were measured using the Cell Death Detection ELISA Plus Kit (Roche Diagnostics) per manufacturer'

s instructions and as previously described [7] with the following change. Absorbance at

405 nm was meas........