Figure 4.

Mcl-1 stability decreased in U373 but not in A172 cells following irradiation. (a) 0–72 h after irradiating A712 and U373 cells with 10 Gy, lysates were made. Phospho (S163)-Mcl-1 and total Mcl-1 were analyzed by western blot. β-actin was used as loading control. A shift of phosphorylated Mcl-1 was observed in U373 cells 48–72 h after irradiation, indicating post-translational modification of Mcl-1. (b, c) 48 h after irradiating A172 and U373 cells with 0 or 10 Gy (IR:- or +), lysates were made. (b) Mcl-1 was precipitated (IP: Mcl-1), and the ubiquitylation status of Mcl-1 was analyzed by western blot. (c) USP9x was precipitated (IP: USP9x), and USP9x as well as co-precipitated Mcl-1 were analyzed by western blot. As a control, precipitation experiments were performed with unspecific isotype-matched immunoglobulins (IP: IgG) and lysates from non-irradiated cells. Ubiquitylated Mcl-1 was increased 48 h after IR in U373 but not in A172 cells. At the same time, more Mcl-1 precipitated with USP9x in A172 cells after irradiation, whereas the association was not changed in U373 cells. Precipitation experiments were performed twice. After 48 h of irradiation with 0 Gy or 10 Gy, A172 (d) and U373 (e) cells were treated with 5 μM cycloheximide (CHX) for the indicated time (upper panels). Cells were lysed at respective time points, and Mcl-1 levels were analyzed by western blot. β-actin was used as loading control. After densitometric quantification, values were normalized to the respective loading control (β-actin) and then to the respective untreated control values. The results of the densitometric analysis are shown below the blots (lower panels). Subsequently, Mcl-1 values were fitted and the half-life of Mcl-1 was calculated (right panels). Bar diagrams show the average half-life time of Mcl-1 in non-irradiated and irradiated cells±S.D. (n=3). Mcl-1 stability remained unchanged in A172 cells but was significantly decreased in U373 cells 48 h after irradiation