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Fig. 6. Restoration of steady-state levels of R14W mutant CA IV protein by chemical chaperones. COS-7 cells transfected with wild-type CA IV (WT) or R14W CA IV (MT) cDNAs were treated with 10 mM ethoxzolamide (Ethox), 10 mM acetazolamide (AC), 5 mM PBA (PBA), or normal medium (Control) for 72 h. The cell lysates containing 5 mg of cell proteins were analyzed by SDS/PAGE under nonreducing conditions followed by Western blot analysis (Upper). The immunoreactive protein concentration of the polypeptides was determined by densitometric scan of the bands. The relative densitometric units were plotted for different chaperones (Lower). The steady-state level of R14W CA IV was restored by ethoxzolamide and acetazolamide. PBA was found to be more effective in restoring the steady-state protein level of mutant CA IV.

Fig. 7. The trans effect of R14W mutant CA IV on the synthesis and secretion of other proteins. COS-7 cells were transfected with vector-only, wild-type CA IV (WT), or R14W CA IV (MT) cDNAs containing human b-glucuronidase (GUS) or secretory CA IV (G267X CA VI) cDNAs. Cell lysates containing 5 mg of cell protein (for GUS) or 5 ml of secretion medium (for G267X CA IV) were analyzed on SDS/PAGE under reducing conditions. The protein polypeptides were characterized by Western blot (Upper). The polypeptide band intensity was determined by densitometric scan, and results are plotted in densitometric units (Lower). In the presence of the R14W CA IV, the expression and secretion of GUS and G267X CA IV were reduced.