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. 2018 Feb 5;14(2):e1007218. doi: 10.1371/journal.pgen.1007218

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© 2018 Li 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 credited.

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Fig 2 — (A-B) The proteasome inhibitor MG132 stabilizes KIX8 (A) and KIX9 (B). Ten-day-old 35S:Myc-KIX8 or 35S:Myc-KIX9 seedlings were incubated with 50 μM MG132 (+) or DMSO control (-) for 0, 4, 8 and 16 hours. Myc-KIX8 and Myc-KIX9 were detected by immunoblot with anti-Myc antibody. Immunoblot analysis using anti-RPN6 antibody was used as loading controls. Two independent lines of 35S:Myc-KIX8 (#2 and #5) and 35S:Myc-KIX9 (#3 and #7) were analyzed. (C-D) The amounts of KIX8 and KIX9 proteins were decreased in plants overexpressing SAP. 35S:Myc-KIX8 (#2 and #5) and 35S:Myc-KIX9 (#3 and #7) transgenic lines were crossed with 35S:GFP (Control) and 35S:GFP-SAP (SAP-OX), respectively. The amounts of Myc-KIX8 or Myc-KIX9 in 35S:GFP;35S:Myc-KIX8 (Control), 35S:GFP-SAP;35S:Myc-KIX8 (SAP-OX), 35S:GFP;35S:Myc-KIX9 (Control), and 35S:GFP-SAP;35S:Myc-KIX9 (SAP-OX) was analyzed with anti-Myc antibody. Immunoblot analysis using anti-RPN6 antibody was used as loading controls. Relative transcription levels of KIX8 and KIX9 in each line were shown at the bottom. (E-F) The amounts of KIX8 and KIX9 proteins were increased in the sod3-1 mutant. Two independent lines of 35S:Myc-KIX8 (#2 and #5) and 35S:Myc-KIX9 (#3 and #7) were crossed with sod3-1, respectively. The amounts of Myc-KIX8 or Myc-KIX9 in 35S: Myc-KIX8, 35S:Myc-KIX8;sod3-1, 35S:Myc-KIX9, 35S:Myc-KIX9;sod3-1 seedlings were analyzed by immunoblot using anti-Myc antibody. Immunoblot analysis using anti-RPN6 antibody was used as loading controls. Relative transcription levels of KIX8 and KIX9 in each line were shown at the bottom. Myc-KIX8 and Myc-KIX9 protein levels in (A) to (F) were quantified by the ImageJ program, and relative levels of Myc-KIX8 and Myc-KIX9 were shown blow the blots. (G) SAP-mediated degradation of PPD is dependent of KIX8/9. Myc-PPD1/2 and GFP-SAP or GFP control were co-expressed in Col-0 or kix8-1 kix9-1 protoplasts, and the amounts of PPD proteins were detected by immunoblot using anti-Myc antibody. EOD1-Flag was used as a control for protoplast transformation. Triplicate transformation evens were performed and representative results were shown. (H) The ppd2-1 mutation did not affect SAP-mediated degradation of KIX8 and KIX9. Myc-KIX8/9 and GFP-SAP or GFP control were co-expressed in Col-0 or ppd2 protoplasts, and the amounts of KIX proteins were detected by immunoblot using anti-Myc antibody. EOD1-Flag was used as a control for protoplast transformation. Triplicate transformation evens were performed and representative results were shown.