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tra12042-sup-0001-FigureS1.docWord document548KFigure S1: The N-terminal c-myc-tag does not influence the localization of KCNE1. Confocal images of WT-KCNE1 transiently expressed in polarized MDCK cells together with the trans-Golgi cisternae and trans-Golgi network marker GalT-mCherry. Inserts in the middle represent zoomed images. WT-KCNE1 displays a localization pattern similar to c-myc-KCNE1 with the strongest signals associated with the trans-Golgi complex. The bottom images are vertical scans of the cells where a minor fraction of WT-KCNE1 can be seen in the apical part of the membrane similar to c-myc-KCNE1. Ap, apical; Ba, basolateral. Scale bars 10 µm.
tra12042-sup-0002-FigureS2.docWord document1255KFigure S2: KCNE1 redistribution to the lateral membranes is KV7.1 specific. A–C) Horizontal and vertical confocal images of polarized MDCK cells transiently expressing KV4.3, KV7.4 or KV11.1 together with c-myc-KCNE1 and co-stained for the F-actin cytoskeleton to indicate the surface membrane. All channels display a primarily basolateral localization and none could relocate KCNE1 to the surface membrane as the subunit is still mainly intracellular in its distribution. Ap, apical; Ba, basolateral. Scale bars 10 µm.
tra12042-sup-0003-FigureS3.docWord document1750KFigure S3: Singly expressed KCNE1 does not change localization during the calcium switch. MDCK cells were transiently transfected with c-myc-KCNE1 and GalT-mCherry, and subjected to a calcium switch assay (see Materials and Methods). The cells were fixed at various time-points (3, 12 and 24 h) after initiation of the polarization process (t = 0 h). The subcellular location of KCNE1 was determined by immunocytochemistry and confocal microscopy. Inserts under each horizontal confocal scan represents a vertical confocal scan of the same cells. KCNE1 did not change localization during the calcium switch as it was primarily associated with GalT at all time-points. Scale bars 10 µm.
tra12042-sup-0004-FigureS4.docWord document1403KFigure S4: Trafficking-deficient KV7.1 mutants are retained in the ER. A–C) Horizontal confocal images of polarized MDCK cells transiently expressing the KV7.1 LQT mutants P117L, E261K and R591H. To visualize the location of the ER, the marker DsRed-ER was coexpressed. KV7.1 LQTS mutations were all strongly associated with the ER and the localization to the cell surface was lost. Scale bars 10 µm.
tra12042-sup-0005-FigureS5.docWord document1210KFigure S5: The LQTS mutant KCNE1-T7I is localized to the Golgi and redirected to the basolateral membrane upon KV7.1 coexpression. N-terminally c-myc-tagged KCNE1-T7I was transiently expressed in polarized MDCK cells and its subcellular localization was investigated using subcellular markers of the ER (DsRed-ER, A) or the trans-Golgi cisternae and network (GalT, B). Phalloidin, which stains F-actin, was included to indicate the localization of the plasma membrane. Confocal microscopy demonstrates that c-myc-KCNE1-T7I displays a primarily intracellular distribution with the strongest staining associated with GalT. C) MDCK cells coexpressing c-myc-KCNE1-T7I and KV7.1. Both subunits were primarily localized in the basolateral membrane. Ap, apical; Ba, basolateral. Scale bars 10 µm.

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