Fig. 6

Rack1 enhances E2F1 stability by inhibiting its ubiquitination. (A) Expression levels of E2F1 mRNA in control and Rack1 knockdown MCF7 and MDA-MB-231 cells. (B) Control and Rack1-silenced MCF7 and MDA-MB-231 cells were incubated with 20 µM cycloheximide (CHX) for the indicated times, and then the protein levels of E2F1 were determined by Western blotting. (C) MCF7 and MDA-MB-231 cells were incubated with CHX (20 µM) or CHX plus the autophagy inhibitor Chloroquine (CQ 200 nM), or MG132 (10 µM) for the indicated times, and then the protein levels of E2F1 were determined by Western blotting. (D-E) Control and Rack1-silenced MCF7 (D) and MDA-MB-231 (E) cells were incubated with CQ (200 nM) or MG132 (10 µM) for 6 h, then the protein levels of E2F1 were determined by Western blotting. (F-G) After transfection of control and Rack1-silenced breast cancer cells with HA-tagged ubiquitin (HA-UB), the cells were treated with MG132 for 6 h and the level of poly-ubiquitinated E2F1 was assessed