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. 2017 Aug 16;7(8):170091. doi: 10.1098/rsob.170091

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© 2017 The Authors.

Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.

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Figure 6. — TatA and TatB cross-linking patterns are altered in the presence of an overproduced Tat substrate. (a) Strain MC4100ΔBC harbouring plasmid p101C*BC producing TatB^L9C alongside TatC^M205C and plasmid pQE80-CueO where indicated, were left untreated (control, C), or incubated with 1.8 mM CuP for 1 min (O). Membrane fractions were prepared, separated by SDS-PAGE (10% polyacrylamide) and immunoblotted with anti-TatB_FL, anti-TatC as indicated. An aliquot of the soluble fraction following membrane preparation was retained and analysed by immunoblotting with an anti-Histag antibody. (b) Strain MC4100ΔBC producing the indicated Cys variants of TatB and TatC from plasmid p101C*BC and his-tagged CueO from pQE80-CueO were incubated with 1.8 mM CuP for 1 min. Following quenching, membrane fractions were separated by SDS-PAGE (10% polyacrylamide) and immunoblotted with an anti-TatC antibody. A non-oxidized sample of membranes harbouring TatB^L9C–TatC^MF213C is shown in the left-most lane. An aliquot of the soluble fraction from each sample was retained and analysed by immunoblotting with an anti-Histag antibody. (c) Whole cells of strain MC4100ΔBC producing TatB^L9C alongside TatC^F213C or TatC^{F94A,E103A,M205C} (annotated TatC^FEA,M205C) from plasmid p101C*BC, and his-tagged CueO (from pQE80-CueO) were incubated for 1 min with 1.8 mM CuP. Following membrane preparation, cross-links were detected with an anti-TatB peptide antibody or an anti-TatC antibody. An aliquot of the soluble fraction from each sample was retained and analysed by immunoblotting with an anti-Histag antibody. (d) Strain DADE harbouring plasmid pTAT101 producing wild-type TatB, TatA^L9C and either TatC^M205C or TatC^F213C along with plasmid pQE80-CueO were left untreated (control, C), or incubated with 1.8 mM CuP for 1 min (O). Membrane fractions were separated by SDS-PAGE (12.5% polyacrylamide) and immunoblotted with an anti-TatC antibody. An aliquot of the soluble fraction from each sample was retained and analysed by immunoblotting with an anti-Histag antibody. p: precursor, m: mature forms of substrate CueO-His * indicates an oxidation product of CueO.