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. 2010 Jan 7;285(9):6145–6152. doi: 10.1074/jbc.M109.062067

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© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 2. — N-terminally tagged A1-chain does not appear to retrotranslocate from ER to cytosol. Retrotranslocation is measured in Vero cells by isolating cytosol from cell membranes and subcellular organelles via centrifugation. The presence of the A1-chain in the cytosolic fraction is analyzed by SDS-PAGE and immunoblot (IB) using antibodies recognizing the A-subunit (CTA) and B-subunit (CTB) or HA tag. A, retrotranslocation results are shown for WT CT (lane 4), the enzymatically inactive E112D mutant (lane 5), and the non-cleavable CT mutant R192G (lane 6). Lanes 1–3 are controls, and lanes 7–9 are the membrane fraction. B, SDS-PAGE and immunoblot of cytosolic and membrane fractions of the retrotranslocation assays using an antibody recognizing ER lumenal marker BiP. C, retrotranslocation assay using WT CT, HA-CT, and CT-HA as indicated. Lanes 1 and 12–14 are controls. * indicates an unidentified band. BFA, brefeldin A.