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Figure 6


Fig. 6. Stability of ADAM22 variant 4 is altered by loss of 14-3-3 binding sites. (A) HEK 293T cells expressing FLAG-tagged ADAM22v4 wild-type or a double substitution mutant of both 14-3-3 consensus binding sites were pre-treated with puromycin to block protein synthesis for 0, 2, 4, 6, or 8 hours, lysed and analysed by western blotting for FLAG-tagged ADAM22 protein levels. This experiment is representative of several previous experiments which yielded similar results. (B) Densitometry of ADAM22 precursor bands from immunoblot normalized to ß-tubulin protein levels and expressed as percentage protein levels of that of wild-type control. (C) Detection of endogenous 14-3-3 isoforms present in D645 and HEK 293T cells. RT-PCR reactions amplifying full length transcripts for 14-3-3 isoforms present in D645 and HEK 293T cDNA. NTC, no template control for 14-3-3{eta} primer set.