Pulse-chase analysis of αi2, sαi2, and αi2A327S Expression vectors encoding αi2, sαi2, or αi2A327 were transfected into COS-7 cells. 48 h after transfection, cells were incubated for 10 min in media containing 0.1 mCi/ml 35S Express protein labeling mix. Cells were washed, and incubated in regular media for the indicated times (chase). Cells were harvested, lysates were prepared, and immunoprecipitations were performed using the EE antibody. Samples were resolved by SDS-PAGE, and radiolabeled proteins were visualized by fluorography (upper panel). Note that two bands are present in the sαi2 lanes; the upper band represents full-length sαi2 and quantitation of this band was used to determine a half-life. The lower band likely represents a degradation product of sαi2 that is itself degraded even faster. Quantitation of the bands from the fluorograph was performed by densitometry and plotted as degradation curves (lower panel) for αi2 (□), sαi2 (O), and αi2A327 (∇). These experiments were repeated and the average ± S.E. half-life was determined as 237 ± 110, 150 ± 42, and 67 ± 21 min for αi2 (n = 3), αi2A327 (n = 2), and sαi2 (n = 3), respectively.
Wedegaertner BMC Cell Biology 2002 3:12 doi:10.1186/1471-2121-3-12