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. 2017 Sep 15;45(20):11891–11907. doi: 10.1093/nar/gkx820

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© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 7. — Plant GSSG treatment affects RTL1 cleavage activity. (A) Top, proteins from Col0 WT and 35S::RTL1 (#1 and #2) plants were extracted and hybridized with α-Flag and α-RTL1 antibodies. Ponceau staining (pink dye) is shown as protein loading control (lanes 1–3 and 4–6). L, indicates protein ladder. Bottom, sequence of dsRNA substrate-2. The putative rcr motif is underlined and the vertical arrows show DCL3/DCL4 cleavage site. (B) [γ-³²P]-ATP RNA substrate-2 was incubated with either WT (lanes 2), 35S:RTL1-Flag #1 (lane 3) or 35S:RTL1-Flag #2 (lane 5) protein extracts or with WT + 35S:RTL1-Flag #1 (lane 4) or WT + 35S:RTL1-Flag #2 (lane 6) protein extracts. Reactions were performed either with 15 μl, 1.5 μl or 15 μl + 1.5 μl. Lane 1 shows [γ-³²P]-ATP RNA substrate-2 only. (C) Glutathione levels in 2-weeks old WT and 35S:RTL1-Flag #1 plantlets treated or not with 10 mM GSSG. Asterisks indicate a significant difference (P ≤ 0.01) between total glutathione levels of WT and 35S:RTL1 plants. Reduced and oxidized glutathione levels are indicated by white and gray bars, respectively. The percentage reduction state of glutathione is indicated above bars. Error bars represent SD (n = 4). (D) [γ-³²P]-ATP RNA substrate-2 was incubated with WT and 35S:RTL1-Flag #1 protein extracts prepared from treated (lanes 3 and 5) or not (lanes 2 and 4) with 10 mM GSSG respectively. Lane 1, shows [γ-³²P]-ATP RNA substrate-2 only and Lane 6, cleavage reaction using 35S:RTL1-Flag #1 protein extract not submitted to GSSG buffer and is used as a positive control.