Figure 10

Pulse-chase analysis of α _i2 , sα _i2 , and α _i2 A327S Expression vectors encoding α_i2, sα_i2, or α_i2A327 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 sα_i2 lanes; the upper band represents full-length sα_i2 and quantitation of this band was used to determine a half-life. The lower band likely represents a degradation product of sα_i2 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 α_i2 (□), sα_i2 (O), and α_i2A327 (∇). These experiments were repeated and the average ± S.E. half-life was determined as 237 ± 110, 150 ± 42, and 67 ± 21 min for α_i2 (n = 3), α_i2A327 (n = 2), and sα_i2 (n = 3), respectively.