FIGURE 7.

Proportion of R-CRT in CRT-EGFP cells. A, stably transfected cells expressing CRT-EGFP and R-CRT-EGFP were treated with MG132 (10 μm) for 12 h to cause accumulation of these proteins. MG132 was removed, and cells were washed 4 times, cultured for 1 h to allow diffusion of the inhibitor, washed again, and incubated for 2 h in presence of CHX. CRT-EGFP and R-CRT-EGFP content was analyzed by immunoblotting using anti-GFP, anti-R-CRT, and anti-β-actin Abs. B, the fraction of R-CRT-EGFP present in CRT-EGFP cells was obtained from the relations between the intensities of EGFP and R-CRT bands and plotted as a percent of the relation between EGFP and R-CRT bands intensities in R-CRT-EGFP cells. The relation between the intensities of EGFP and R-CRT bands in R-CRT-EGFP cells reflects the different sensitivity of anti-GFP and anti-R-CRT Abs and corresponds to 100% of R-CRT-EGFP. The estimated non-arginylated CRT-EGFP in CRT-EGFP cells was 40%, corresponding to the point 2 h in the half-life determination experiments (Fig. 5). Data represent the mean ± S.E. of seven independent experiments.