Figure 1.

Staurosporine (STS) induces selective degradation of MIEF1 via the ubiquitin-proteasome pathway. (a) HeLa cells were treated with DMSO, 100 nM STS, 0.3 µM actinomycin D (ActD) or 20 ng/ml tumor necrosis factor (TNF) plus 3 μg/ml cycloheximide (CHX) for 16 h. Mitochondrial fractions of cells were then subjected to immunoblotting. Asterisks indicate non-specific bands. (b) Quantification of the levels of MIEF2 and MIEF1 in (a) is shown. Averages are shown as quantified by densitometry from 3 independent experiments. The levels of MIEF2 or MIEF1 proteins were normalized to HSPD1. Error bars represent standard deviation (SD). ***P < 0.001, NS, not significant. (c) Mitochondrial fractions of U2OS cells treated as indicated were analyzed by immunoblotting. (d) The levels of MIEF1 in U2OS cells were quantified by densitometry from 3 independent experiments. Bars indicate mean ± SD. ***P < 0.001, NS, not significant. (e) HeLa cells were transiently transfected with Flag-tagged vector or MIEF1 and MYC-tagged Ubiquitin for 24 h. After treatment with 100 nM STS and 1 μM MG132 for 16 h, cells were lysed and subjected to immunoprecipitation (IP) with anti-Flag antibody. IP products and cell lysates were detected with anti-Flag and anti-Ub. Asterisks indicate non-specific bands. (f) Mitochondrial fractions of HeLa cells treated as indicated were subjected to SDS-PAGE. (g) MIEF1 level normalized to the HSPD1 expression is presented. Bar chart represents data from 3 independent experiments as in (f). Error bars indicate SD. ***P < 0.001, NS, not significant.