Fig. 7. Inhibition of the proteasome leads to an increase in mutant opsin. Western blot using mAb 1D4 of opsin in DM-soluble (S) and DM-insoluble (P) protein extracts showed that inhibition of the proteasome machinery with MG-132 (5 µM for 16 hours) (+) led to an increase in the steady state level of mutant opsin in COS-7 cells. The level of WT protein (WT-opsin) was not affected by proteasome inhibition, whereas the level of mutant proteins (P23H-opsin and K296E-opsin) showed a dramatic increase. The electrophoretic mobilities of different glycoforms of opsin were determined empirically using PNGase F and Endo H: mature (40 kDa) (arrowhead); and immature forms (>41 kDa); deglycosylated form (30 kDa) (^*) and dimer (60 kDa) (^**). Blot exposures have been adjusted to give equivalent band intensity between WT and mutants, as the WT protein is far more abundant. The positions of the molecular weight markers are indicated on the left in kDa.

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