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. 2012 May 17;8(5):e1002707. doi: 10.1371/journal.ppat.1002707

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Xin et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Purification and immunoblot analysis of His-tagged NopM and point-mutated His-tagged NopM-C338A (marked by an arrow). Proteins purified by nickel–nitrilotriacetic acid affinity chromatography were obtained from E. coli BL21 (DE3) harboring pET-nopM (lane NopM) and pET-nopM(C338A) (lane C338A), respectively. BL21 (DE3) with the empty vector pET28b was used as a control (lane EV). The SDS-PAGE gel (0.5 µg loaded proteins) was stained with Coomassie Brilliant Blue R-250 and the corresponding immunoblot with the rabbit serum raised against NopM was developed with 3, 3′-diamino-benzidine. (B) In vitro ubiquitination reactions with indicated purified proteins followed by immunoblot analysis with chemiluminescence reagents using anti-HA and anti-NopM antibodies. Ubiquitination reactions were performed in the presence or absence of HA-tagged ubiquitin, E1, UbcH5B, His-tagged NopM and His-tagged NopM-C338A for 1 h at 37°C.