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Fig. 6. The N-terminal domain bears a cleavable signal sequence and confers instability to hFBNRG3. (A) Attachment of the HA epitope to the N-terminal domain of hFBNRG3-myc produced no immunoreactivity in anti-HA immunoblots (lane 4) even in the proteasome inhibited cells (lane 3), whereas an HA-tagged control protein was clearly detected (lane 2). As expected, HA immunoreactivity was not detected in non-HA-tagged hFBNRG3 (lanes 5 and 6) and nontransfected cells (lane 1). The band of 
90 kDa present in the immunoblots is unspecific as demonstrated by its presence in the nontransfected cells. (B) HA-hFBNRG3-myc was expressed at levels comparable with those of hFBNRG3-myc as demonstrated by the anti-myc immunoblot of aliquots of the same cell extracts. (C) Protein stability is increased when the peptide harbouring the signal sequence is deleted (lane 3). The attachment of the peptide to the transmembrane protein synaptotagmin I downregulates its expression levels in transfected cells (lane 5). (D) N-terminal domain bearing CFP chimeras (lanes 1, 3 and 5) were hardly detectable in anti-GFP immunoblots, whereas peptide-deleted chimeras (lanes 2 and 4) showed a considerable increase in protein levels. COS-7 cells were transfected with the indicated vectors and cell extracts were immunoblotted with anti-myc, anti-HA or anti-GFP antibody.
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