A) COS-7 cells were treated with MG132 (50 μM) for times indicated. Equal amounts of protein (50 μg) were separated on 7.5% SDS-PAGE gel, transferred to PVDF membrane, and detected with an eIF-4G polyclonal antibody as described in Experimental Procedures. B) To detect phosphorylated eIF2α and total eIF2α, equal amounts of protein (50μg) were separated on 12.5% SDS-PAGE gel, followed by Western blot analysis as described in Experimental Procedures. C) The results were quantitated by ImageQuant software, and the data is expressed as the level of phosphorylated eIF2α divided by the total level of eIF2α, with time point 0 set as one. Values are means±SEM (n = 2). The error bars are small and not visible for some data points.