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Fig. 4. The ubiquitin/proteasome system regulates the protein stability of hFBNRG3. (A) Proteasome inhibition allows the detection of hFBNRG3 protein expression in anti-myc western blots of transiently transfected COS-7 cells. Nontreated cells (lane 5) showed no immunoreactivity at these conditions. Treatment with three different proteasome inhibitors Lactacystin, Z-Leu-Leu-Phe-CHO (Z-L-L-F-al) and MG-132 (lanes 6, 7 and 8, respectively) resulted in the appearance of immunoreactivity. The transfection of the inversely oriented cDNA produced no immunoreactivity (lane 1), even in the presence of the proteasome inhibitors (lanes 2, 3 and 4). Similar results were obtained in the neuron-like PC12 cells (lanes 9 to 12). Although a faint band was detected in nontreated PC 12 cells (lane 12) it was shown to be unspecific because it was also present in cells transfected with inversely oriented cDNA. (B) Time course of protein stabilization after proteasome inhibition with 2 µM MG-132 in COS-7 cells. As shown, immunoreactivity was already detected after 4 hours of proteasome inhibition. (C) Impairment of the ubiquitylation machinery by incubating the cells at 42°C increased the protein stability in the mammalian conditional mutant cell line ts20. An unspecific band of 
65 kDa was detected at 42°C even in cells that had been transfected with inversely oriented cDNA.
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