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. 2018 Jul 13;9:930. doi: 10.3389/fpls.2018.00930

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Copyright © 2018 Hussain, Sheikh, Haider, Quareshy and Linthorst.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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AtWRKY50 and TGA2 and TGA5 bind to the PR1 promoter. (A) EMSAs were performed with an 80-bp fragment of the PR1 promoter without protein (minus signs) or with GST-tagged C-terminal half (C) or full-length (FL) versions of AtWRKY50, and His-tagged TGA2 (T2) or TGA5 (T5), and combinations of these proteins, as indicated above the lanes. The positions of the unbound probe (FP), the top of the gel (Top), and of band shifts caused by one (1W) or two (2W) AtWRKY50-C proteins are indicated at the left. Shifts caused by binding of single (single black asterisks) or multiple (single white asterisks) TGA proteins and shifts caused by a combination of TGA and AtWRKY50 (double black asterisks) are indicated. (B) Sequences of PR1 promoter fragments used for electromobility shift assays. Overlapping subfragments A, B, C, and D, and subfragments ABC and BCD are aligned with the sequence of the 80-bp fragment. The overlapping W-box (TTGACT) and AtWRKY50 binding sequence (GACTGTTTC) in LS4, the CGTCA boxes of the as-1 element in LS5 and LS7, and the AtWRKY50 binding sequence (GACTTTTC) in LS10 are indicated in bold. Subfragments ABCm1, ABCm2, BCDm1 and BCDm2 represent variants of fragments ABC and BCD with mutations (underlined) in the CGTCA boxes in LS5 and LS7, respectively. (C) EMSAs were performed with probes corresponding to PR1 promoter fragments ABC and BCD and their mutated versions ABCm1, ABCm2, BCDm1, BCDm2, as indicated above the panels. EMSA incubation mixtures contained no protein (Lanes 1), AtWRKY50-C (Lanes 2), TGA2 (Lanes 3), AtWRKY50-C and TGA2 (Lanes 4), full-length AtWRKY50 (Lanes 5), and full-length AtWRKY50 and TGA2 (Lanes 6). The positions of the unbound probe (FP) and of band shifts caused by AtWRKY50-C (1W) or TGA2 (1T) are indicated at the left. Shifts caused by binding of a combination of TGA and AtWRKY50 (double black asterisks) are indicated. (D) EMSAs were performed with PR1 promoter fragments A, B, C, and D as probes, as indicated above the panels. EMSA incubation mixtures contained no protein (Lanes 1), AtWRKY50-C (Lanes 2), TGA2 (Lanes 3), AtWRKY50-C and TGA2 (Lanes 4), full-length AtWRKY50 (Lanes 5), and full-length AtWRKY50 and TGA2 (Lanes 6). The positions of the unbound probe (FP), the top of the gel (Top) and of band shifts caused by AtWRKY50-C (1W) or TGA2 (1T) are indicated at the left.