Figure 4.

Elevated levels of TPPII enable EL-4 cells to survive toxic concentrations of NLVS and process Ub-conjugated proteins. (A) Relative TPPII activity. FP-biotin binds TPPII at its active site in EL-4, TPPII transfectants, and adapted cells. The 60-kDa band is an unidentified polypeptide detected by Streptavidin-horseradish peroxidase. (B) Viability of EL-4, adapted, and TPPII transfectant cells treated with 0 (squares), 50 (rectangles), and 100 μM (circles) NLVS over 4 days as monitored by flow cytometry. (C) Western blot of accumulated poly-Ub-conjugated proteins using lysates from EL-4, TPPII transfectant, and adapted cells at 0, 4, 8, and 24 h after treatment with 50 μM NLVS assayed for an accumulation of polyubiquitinated proteins as described in Materials and Methods. Less polyubiquinated material is observed after 24 h, the result of partial cell death. One of 10 experiments is shown. (D) Pulse–chase analysis of polyubiquitinated material: control EL-4 (squares), adapted (circles), and TPPII transfectant cells (rectangles) were pulsed with [³⁵S]methionine and 50 μM NLVS and chased over time. Ub conjugates were analyzed by immunoprecipitation, SDS/PAGE, and autoradiography. Lanes were quantified by densitometric scan, and standard error mean was calculated. (E) NLVS is not a substrate for TPPII. EL-4 cells transfected with TPPII were either untreated or incubated with 50 μM NLVS for 24 h at 37°C. Lysates prepared from EL-4, TPPII transfectants, and adapted EL-4 cells were incubated with ¹²⁵I-NLVS as described above.