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. 2017 Aug 25;7:9470. doi: 10.1038/s41598-017-09941-4

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© The Author(s) 2017

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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ATPase enzyme inhibition and tau-phosphorylation assay of MARK4. (A,B) Shows the hydrolysis of Pi from ATP, position of Pi and ATP spots are indicated on left side. Lane 1, negative control (without protein); lane 2, 100 nM MARK4 (positive control); and lanes labeled as 10,20,40,60 and 80, shows the concentration of rutin and vanillin in µM incubated with 100 ng MARK4, respectively. (C,D) ATPase inhibition (% hydrolysis of ATP) with increasing concentrations of rutin and vanillin are shown in bar diagram. Bar graph represents the normalized intensity of ATP hydrolysis for duplicate measurements ± SD. *p < 0.05; **p < 0.01, compared with the control. Statistical analysis was done using Student’s t- test for unpaired samples. (E) Representative flow cytometry histogram of SH-SY5Y cell fractions stained with phosphorylated anti-tau antibodies, each histogram represents the phosphorylation status of tau under different treatments as mentioned in inset (F) NXN plot of SH-SY5Y cells treated with rutin and vanillin stained with phosphorylated anti-tau. It represents the multiple parametric analysis of cell fractions under different treatments stained with phosphorylated anti-tau.