FIG. 6.
The amino acid 435 to 457 region within the C terminal of wt Cot confers susceptibility to proteasome degradation and is independent of PKB activity. (A) HEK293 cells were cotransfected with 5 μg of EYFP-Cot390-467 together with 15 μg of HA-PKB-DD or empty vector, and 14.5 h after transfection the cells were further incubated with 100 μg of cycloheximide/ml in the presence or absence of 10 μM lactacystin as described in Materials and Methods. The figure is representative of the three experiments performed. (B) Two hours after the transfection of HEK293 cells with 10 μg of EYFP-Cot390-467, the cells were incubated with 50 μM LY 294002 and the medium was then supplemented with 100 μg of cycloheximide/ml and 50 μM LY 294002, with and without 10 μM lactacystin, for a further 8 h. The figure shows representative results of the three experiments performed. (C) HEK293 cells were transfected with 10 μg of pEYFP-Cot390-467, pEYFP-Cot390-457, pEYFP-Cot390-446, pEYFP-Cot390-435, or pEYFP-Cot390-424, and after 14.5 h cells were treated as described in the legend for Fig. 5B. Total extracts were Western blotted and probed with anti-EYFP and anti-PDI antibodies; the figure shows representative results of one of the three experiments. (D) HEK293 cells transfected for 14.5 h with pEYFP-Cot414-457, pEYFP-Cot426-457, pEYFP-Cot435-457, and pEYFP-Cot445-450 were treated as explained above for panel C. The figure shows one of the two experiments performed in duplicate.