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The small difference between the mutated constructs VPAC1-C and VPAC1-T was not significant.
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All plasmid constructs were confirmed by DNA sequencing and in all cases the nucleotide substitution introduced represented the only difference between the mutated construct and its corresponding normal control.
The mutated constructs were sequence verified prior to the interaction studies.
The expression pattern of the mutated constructs was not changed in comparison to their wild-type counterparts.
Furthermore, the biological response to bTSH of the cells transfected with the DNA of the mutated constructs was explored in terms of cAMP accumulation.
The mutated constructs were confirmed by DNA sequencing.
Neither of the mutated constructs exhibited increased protein levels upon calyculin A treatment.
One outstanding characteristic of the cells transfected with the mutated constructs was the abundance of filopodia-like cytoplasmic processes.
None of the mutated constructs showed a marked reduction or even absence of pTRY-A3, B GUS expression.
SH-SY5Y cells were transiently transfected with the mutated constructs and analyzed by fluorescence microscopy for PDI subcellular localization.
The mutated construct was generated by using PCR-based mutagenesis of the 1014 bp VPAC1 3'UTR and the primers: 5'-agtgggttattcgtcagtttttgtttggag-3' and 5'ctccaaacaaaaactgacgaataacccact-3'.
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