Figure 3. Expression, stability, and pro-survival effects of proteasome-resistant YB-1 mutants in cells treated with doxorubicin. (A) NIH3T3 cells were transfected with plasmids encoding YB-1 mutants, as indicated, and 24 h later analyzed by WB using YB-1 and β-tubulin antibodies. (B) Cells were transfected as in (A) and then left untreated or treated with 5 µM doxorubicin for additional 14 h. Whole-cell extracts were analyzed by WB. YB-1 was detected using HA antibodies. The numbers at the bottom indicate averaged percentage of the remaining protein as measured from 3 independent experiments by densitometry. β-tubulin was used as a loading control, and PABP was monitored as an additional control for protein stability. (C) Effect of YB-1 mutants on NIH3T3 cell survival after treatment with 5 µM doxorubicin for 14 h was monitored by trypan blue staining (t test, P value [**] <0.01).