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. 2018 Jul 6;8:10279. doi: 10.1038/s41598-018-28188-1

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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In vivo phosphorylation of PIN3-YFP and mutant variants. (a) Transiently expressed PIN3-YFP and all mutant variants in N. benthamiana with or without co-infiltration with PINOID-FLAG. Total protein was extracted, separated by SDS-PAGE, blotted and probed with anti-GFP and anti-FLAG. Phosphorylation of PIN3-YFP, PIN3-YFP-P2A and PIN3-YFP-P2D by PINOID appears as a distinct smear above the main band (marked with star). Results from one single blot are shown, some lines were exposed longer due to weaker signals. The complete blots and Ponceau stain are shown in Figure S3. N indicates carryover signal from anti-GFP (breakdown product of PIN3-YFP-P1D). (b) Lane profiles of the anti-GFP blots for PIN3-YFP and PIN3-YFP-P1A with or without PINOID from (a). Arrow marks additional peak representing phosphorylated protein. (c) Faster migration of PIN3-YFP-P1A and PIN3-YFP-P1D in SDS-PAGE compared to WT and PIN3-YFP-P2A/D (d) Phostag gel revealed no enhancement in the differences of the migration between PIN3-YFP and PIN3-YFP-P1A. (e) Rootward transport of radiolabeled ³H-IAA in decapitated hypocotyls. Neg represents negative control for diffusion in agar. Treatment of 10 μM N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, was used as additional negative control. Student’s T-test was calculated for the comparison of each line with the control (Col-0). Error bars represent SE.