(Downloading may take up to 30 seconds.
If the slide opens in your browser, select File -> Save As to save it.)
Click on image to view larger version.


Figure 4


Fig. 4. Up- and downregulation of MLN51 in HeLa cells. (A) Western blot containing ~20 µg of total protein obtained from: HeLa Tet-On control cells (lanes 1 and 2) stably transfected with the empty pUHD vector; HeLa Tet-On MLN51Fl (lanes 3 and 4) and HeLa Tet-On MLN51Nt (lanes 5 and 6) cells, transfected with pUHD-MLN51/1-703 and pUHD-MLN51/1-383, respectively. The blot was probed with an anti-MLN51 against the SELOR domain of MLN51 and then probed with an anti-tubulin antibody which was used as a loading control. A 24-hour induction with doxycyclin is indicated on the top (+dox). The Arrow and arrowhead on the right indicate the position of MLN51Fl and MLN51Nt, respectively. (B) Western blot analysis of MLN51 depletion in HeLa cells. Western blot containing 20 µg of total protein obtained from HeLa cells transfected twice with either synthetic control siRNA (lanes 1 and 3) or MLN51-specific siRNA (lanes 2 and 4), and with pEYFP-MLN51ins plasmid was probed with anti-MLN51 and anti-tubulin (loading control) antibodies. The positions of the endogenous MLN51 and of the EYFP-MLN51 fusion protein are indicated on the right. (C) Analysis of arsenite-induced EIF2{alpha} (EIF2{alpha}) phosphorylation in the presence of MLN51Fl and MLN51Nt. 20 µg of total protein obtained from doxycyclin-induced HeLa Tet-On control (lanes 1 and 2); HeLa Tet-On MLN51Fl (lanes 3 and 4) and HeLa Tet-On MLN51Nt (lanes 5 and 6) cells, treated (+) or not (–) with arsenite, were analysed by western blotting using anti-MLN51-, anti-EIF2{alpha}-, anti-phospho-EIF2{alpha}- and anti-tubulin-specific antibodies. Arrow and arrowhead on the right indicate the position of MLN51Fl and MLN51Nt, respectively. (D) Analysis of arsenite-induced EIF2{alpha} phosphorylation in MLN51-silenced cells. 20 µg of total protein obtained from HeLa cells transfected twice with either synthetic control siRNA (lanes 1 and 2) or MLN51-specific siRNA (lanes 3 and 4), treated (+) or not (–) with arsenite, were analysed by western blotting using anti-MLN51-, anti-EIF2{alpha}-, anti-phospho-EIF2{alpha}- and anti-tubulin-specific antibodies. (E) Analysis of EIF2{alpha} phosphorylation in HeLa cells treated with arsenite or hippuristanol. 20 µg of total protein obtained from HeLa cells untreated (–, lane 1), treated with arsenite (A, lane 2) or with hippuristanol (H, lane 3) were analysed by western blotting using anti-EIF2{alpha}-, anti-phospho-EIF2{alpha}- and anti-tubulin-specific antibodies.