Fig. 5:

Effect of mutations in Bim that prevent ubiquitylation. (A) Phase contrast (a, c, & e) and immunofluorescence microscopy.: bim-/- bone marrow cells cultured in the presence of M-CSF were infected with either pMx-IRES-EGFP, pMxBimEL-IRES-EGFP or pMxmtBimEL-IRES-EGFP: zVAD-FMK was added just after the retroviral infection. After 7 days of the retrovirus infection, when gene expression was confirmed by EGFP fluorescence (b, d, & f), cultures were deprived of zVAD-FMK. Eighteen hours after zVAD-FMK removal, more than 70% of pMx-IRES-EGFP- and pMxBimEL-IRES-EGFP-infected cells survived as identified by EGFP fluorescence (h and j), compared to the survival rate of 5% in pMxmtBimEL-IRES-EGFP virus-infected cells (l). Both pMxBimEL-IRES-EGFP- or pMxmtBimEL-IRES-EGFP-infected cells died when M-CSF was removed from the cultures (i & k). Bar: 50 μm. (B) The survival ratio of EGFP positive cells. Almost 100% of the control cells survived even 18h after z-VAD-FMK removal in the presence of M-CSF. 18 hours after zVAD-FMK removal, more than 70% of pMx-IRES-EGFP- and pMxBimEL-IRES-EGFP-infected cells survived as identified by EGFP fluorescence (EGFP and wtBimEL), compared to the survival rate of 5% in pMxmtBimEL-IRES-EGFP virus-infected cells (mtBimEL). (C) The proteasome inhibitors MG132 enhanced the expression level of wtBim in pMxBimEL-IRES-EGFP-infected OC precursors even in the presence of M-CSF, while no obvious upregulation of mtBim was observed in pMxmtBimEL-IRES-EGFP infected cells. (D) WtBim or mtBim was immunoprecipitated from cell lysates of pMxBimEL-IRES-EGFP-infected or pMxmtBimEL-IRES-EGFP-infected cells using anti-Bim polyclonal antibody, and the immunoprecipitates were immunoblotted with anti-ubiquitin antibody. Treating the cells with the proteasome inhibitor MG132 strongly increased the ubiquitination of Bim, while no ubiquitination of mtBim was observed. (Ref. 15; Copyright 2003. The European Molecular Biology Organization).