Figure 7.

Interaction of Elongin BC with SOCS-1 increases its stability. 293T cells were transiently transfected in 35-mm dishes with 1 μg of the HPC4-Elongin B expression vector, 1 μg of the HSV-Elongin C expression vector, and 1 μg of the wild-type T7-SOCS-1 expression vector or 3 μg of the T7-SOCS-1 M1 expression vector as indicated. (A) Cycloheximide block. Twenty-four hours after transfection, 100 μg/ml cycloheximide was added to culture dishes. Cells were harvested and lysed after an additional incubation for the indicated times. Twenty-five micrograms of total cell lysate protein was immunoblotted with the indicated antibodies. SOCS-1 protein was quantitated by densitometry of Western blots using NIH Image 1.60/ppc. (B) [³⁵S]methionine pulse chase. Cultures were washed twice with PBS and incubated with methionine-free medium containing 10% dialyzed fetal calf serum for 1 hr prior to addition of [³⁵S]methionine (0.5 mCi/ml of medium; EXPRE35S35S protein labeling mix, NEN), followed by an additional 2 hr incubation. The cells were then washed twice with PBS and cultured in DMEM with 10% fetal calf serum. Cells were harvested and lysed after an additional incubation for the indicated times. Approximately 200 μg of total cell lysate protein was immunoprecipitated with T7 antibody and analyzed by SDS-PAGE. Radioactive proteins were detected and quantitated by PhosphorImager analysis. The data plotted represents the mean of three independent experiments. Error bars indicate the standard error of the mean.