Figure 9. Cystatin A inhibits the processing of MYOC wild-type in cultured cells.

Recombinant expression plasmids containing tag-fused full coding wild-type MYOC (pMG29), CSTA, and controls plasmids, inactive mutated CSTA (CSTAm) and pEmpty, were generated as indicated in Methods. pMG29 was co-transfected with either pCSTA, pCSTAm or pEmpty (1∶2) and harvested at 48 h post-transfection. Equivalent volumes of cell extracts and of their supernatants were loaded onto 4–15% SDS-PAGE gels, transferred to PVDF membranes and analyzed by immunoblotting. Different MYOC protein forms (full length and processed) were detected with an anti-V5 mouse monoclonal followed by an anti-mouse horseradish peroxidase antibodies. Blots were re-probed with β-actin and DDK antibodies for loading and identification controls. Percent of the MYOC processed band was calculated by densitometry. A) schematic representation of the expression cassettes of the recombinant plasmids. B, C and D: Representative western blots with extracts from transfected cells. B) extracts from HEK293 co-transfected by calcium phosphate. C and D) extracts from primary HTM-137 cells co-transfected by nucleofector electroporation.