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Effects of PDRG1 in DNA methylation and MAT activity.

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posted on 22.08.2016 by Claudia Pérez, Francisco J. Pérez-Zúñiga, Francisco Garrido, Edel Reytor, Francisco Portillo, María A. Pajares

(A) Global DNA methylation levels of CHO cells transiently transfected with pHA-PDRG1, pFLAG-MAT or both plasmids evaluated with the inverse radioactive assay and compared to mock transfected cells. Incorporation of methyl groups into DNA (mean ± SEM) of five independent experiments carried out in triplicate is shown. For graphical purposes, the data are expressed as percentage of the pFLAG control taken as 100% (23392.65 ± 1790.07 cpm). Statistical analysis was performed using GraphPad Prism and changes were considered significant when p≤0.05 (*vs. pFLAG; ** vs. pHA; ***vs.FLAG-MAT). (B) Purified recombinant MATα1 (0.7 μM) was incubated with 0–5.6 μM PDRG1 (black) and S-adenosylmethionine synthesis determined; the panel shows results (mean ± SEM) of a typical experiment out of five carried out in triplicate. Controls including MATα1 and histone IIA (red) were also performed (C) Results (mean ± SEM) of a typical activity assay out of three performed in triplicate using MATα2 (0.7 μM). (D) Effects of PDRG1 (mean ± SEM) on MAT II activity (0.7 μM) from a typical experiment out of three carried out in triplicate. (E) Typical profile of a Biogel A purification of the MATα1/GST-PDRG1 complex followed by MAT activity. Elution of the standards is indicated with sticks that correspond to: Blue dextran (40 ml); ferritin (48 ml); aldolase (69 ml); conalbumin (81 ml); ovalbumin (84 ml); and ATP (105 ml). The upper part of the panel shows a stained SDS-PAGE gel of the relevant fractions as indicated on the top; the molecular size of the markers shown in the last lane (right) is indicated next to the corresponding stained band. (F) Comparison of the MAT activity shown by the MATα1/GST-PDRG1 (MATα1-HE; top) and MATα2/GST-PDRG1 complexes (MATα2-HE; bottom) vs. MATα1 or MATα2 homo-oligomers as correspond. The results shown are mean ± SEM of three independent experiments; *p<0.05.