FIGURE 10.
Studying protein degradation by the proteasome under controlled cellular conditions. PC12 stably transfected with EGFPd2 were treated with MG132 (5 μm) for 4 h, then washed four times with complete medium to remove the excess proteasome inhibitor, and plated again. 1 h after plating medium was changed, and fresh media containing no addition (control), 20 μg/ml CHX, or CHX and proteasome inhibitors as indicated in the figure were added. The left panel shows the quantification of relative fluorescence and protein levels as determined by flow cytometry and Western immunoblot. Data are expressed as mean ± S.E. from three different experiments. The middle panel shows a representative Western and immunoblot experiment of proteins (EGFPd2 and tubulin, which was used as control) immunoblotted with anti-EGFP and anti-tubulin antibodies. Φ, untransfected PC12. Right panel, total RNA was isolated and analyzed by qPCR as described under “Experimental Procedures.” Graphs show the relative -fold change and the corresponding ranges (upper and lower bars) as calculated from three different experiments from −ΔΔCT values. Lacta, lactacystin.