GST-RTA(N)-WT or GST-RTA(N)-DD was expressed and purified from E. coli strain BL21. FLAG-tagged PFAS or PFAS-ED was purified from transfected 293T cells. In vitro on-column deamidation of RTA(N) was performed as previously reported (9, 10). Briefly, 1 μg of PFAS/PFAS-ED and 0.5 μg of GST-RTA(N) (immobilized on glutathione-conjugated agarose) were added to a total volume of 50 μl. The reaction was carried out at 37°C for 45 min in deamidation buffer [50 mM tris-HCl (pH 7.5), 100 mM NaCl, and 5 mM MgCl2]. Protein-bound GST beads were washed with deamidation buffer, and GST-RTA(N) was eluted with rehydration buffer (8 M urea, 2 M thiourea, 2% CHAPS, 0.5% IPG buffer, and 0.002% bromophenol blue) at room temperature. Samples were then analyzed by two-dimensional gel electrophoresis and immunoblotting.

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