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. 2018 Jul 18;293(36):13932–13945. doi: 10.1074/jbc.RA118.004183

Figure 2.

Figure 2.

EDEM1 possesses ∼4 reactive thiols. A, EDEM1-FLAG was expressed in HEK293T cells. Cells were pretreated with DTT (5 mm) where indicated (+/−). Cells were lysed in sample buffer containing 5 mm PEG-maleimide (+PEG-maleimide) or 20 mm NEM (−PEG-maleimide). Proteins were resolved by 8.5% SDS-PAGE and immunoblotted (IB) with FLAG antibody. B, structural model (Phyre2.0) of the MLD (cyan) showing the three catalytic triad (red) and Cys residues (yellow). The cartoon representation of EDEM1 depicts signal peptide (green), MLD (cyan), putative catalytic residues (red), Cys residues (yellow), and predicted disulfides (*). C, FLAG-tagged EDEM1 (WT), C95S/C302S, C555S/C629S, C95S/C302S/C629S, and C95S/C302S/C555S/C629S were expressed in HEK293T cells and treated as in A. D, FLAG-tagged EDEM1 (WT), C95S/C302S/C629S, and C95S/C302S/C555S/C629S were coexpressed with A1AT WT, Z, and NHK in cells. Cells were radiolabeled with [35S]Cys/Met for 30 min and chased for 1 h. Cells were lysed in MNT buffer containing 0.1% SDS. Half of the cell lysate was subjected to anti-A1AT, and half was subjected to anti-FLAG immunoprecipitation. Proteins were resolved by 8.5% reducing SDS-PAGE. The asterisk denotes background bands. Gels are representative of three independent experiments.