10.1371/journal.pntd.0008458.g002 Juan F. Quintana Juan F. Quintana Juan Bueren-Calabuig Juan Bueren-Calabuig Fabio Zuccotto Fabio Zuccotto Harry P. de Koning Harry P. de Koning David Horn David Horn Mark C. Field Mark C. Field Characterisation of tagged TbAQP2. Public Library of Science 2020 TbAQP 2 stability ER cytoplasmic-oriented lysine residues drug resistance ubiquitin conjugation sites TbAQP 2 TbAQP 2 mediates pentamidine transl... flagellar pocket membrane TbAQP 2 mediates pentamidine sensit... 2020-07-09 17:54:34 Figure https://plos.figshare.com/articles/figure/Characterisation_of_tagged_TbAQP2_/12635405 <p><b>A)</b> Fluorescence microscopy of <i>T</i>. <i>brucei</i> 2T1 cells expressing tetracycline-regulated N- or C-terminal tagged AQP2 (<sup>3xHA</sup>AQP2 or AQP2<sup>3xHA</sup>, respectively, in yellow). These proteins localise similar to ISG75 (magenta) at the flagellar pocket/endosomes. The triple <i>aqp-</i>null <i>T</i>. <i>brucei</i> 2T1 cells (ΔAQP) were also included as control. Scale bar 5 μm. <b>B)</b> Tet-regulated expression of N- or C-terminal HA-tagged AQP2. Both native-PAGE (upper panel) and SDS-PAGE (lower panel) αHA blots are shown. α–β tubulin was used as loading control. The presence of the different oligomeric species is indicated in the right-hand side of the panel. Note the presence of a high molecular weight form under SDS-PAGE in <sup>3xHA</sup>AQP2 but not AQP2<sup>3xHA</sup>. The triple <i>aqp-</i>null <i>T</i>. <i>brucei</i> 2T1 cells (ΔAQP) were also included as control. <b>C)</b> EC<sub>50</sub> values for pentamidine <b>(left panel)</b> or salicylhydroxamic acid (SHAM; <b>right panel</b>) with or without 5 mM glycerol following expression of either <sup>3xHA</sup>AQP2 or AQP2<sup>3xHA</sup>. For multiparametric ANOVA, we compared the average values (n = 4 independent replicates) from wild type <i>T</i>. <i>brucei</i> 2T1 cells as reference for pentamidine, or from <i>aqp-</i>null cells for SHAM. * <i>p</i><0.01, ** <i>p</i><0.001, *** <i>p</i><0.0001 from four independent replicates. <b>D) Left panel;</b> Representative western blotting (n = 3 independent replicates) from protein turnover assay monitored by cycloheximide (CHX) treatment in <i>T</i>. <i>brucei</i> 2T1 cells expressing either <sup>3xHA</sup>AQP2 (upper panel) or AQP2<sup>3xHA</sup> (lower panel). <b>Right panel</b>; Protein quantification from western blotting analysis in left panel for either <sup>3xHA</sup>AQP2 (black square) or AQP2<sup>3xHA</sup> (grey circles). Results are the mean ± standard deviation of three independent experiments (n = 3 independent replicates). The estimated half-life (t<sub>1/2</sub>) was calculated based on regression analysis using PRISM.</p>