Open Access Research article

A human polymorphism affects NEDD4L subcellular targeting by leading to two isoforms that contain or lack a C2 domain

Nicholas F Garrone1, Bonnie L Blazer-Yost2, Robert B Weiss1, Jean-Marc Lalouel1 and Andreas Rohrwasser1*

Author Affiliations

1 Department of Human Genetics, Eccles Institute of Human Genetics, University of Utah School of Medicine, Salt Lake City, USA

2 Department of Biology, Indiana University Purdue University at Indianapolis, Indianapolis, USA

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BMC Cell Biology 2009, 10:26  doi:10.1186/1471-2121-10-26

Published: 13 April 2009

Additional files

Additional file 1:

EGFP-NEDD4L-C2(-) does not colocalize with markers of mitochondria, the endoplasmic reticulum (ER), the Golgi complex or lysosome. Confocal images of live A6 cells that were transiently transfected with EGFP-NEDD4L-C2(-) (A-L) and incubated in the presence of MitoTracker® Red (A-C), ER-Tracker™ Red (D-F), BODIPY® TR C5-ceramide complexed to BSA (Golgi marker) (G-I) or Lysotracker® Red (J-L). Blue and green channel overlay (A, D, G, J). Blue and red channel overlay (B, E, H, K). Blue, green and red channel overlay (C, F, I, L). Scale bars are equivalent to 10 μm.

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Additional file 2:

In response to a Ca2+ stimulus, EGFP-NEDD4L-C2(+) does not target the endoplasmic reticulum, lysosome, Golgi complex, mitochondria, early endosome or autophagosome. Confocal images of live A6 cells transiently transfected with EGFP-NEDD4L-C2(+), treated with 10 uM ionomycin and 1.97 mM Ca2+, and incubated in the presence of ER-Tracker™ Red (A), Lysotracker® Red (B), BODIPY® TR C5-ceramide complexed to BSA (Golgi marker) (C), MitoTracker® Red (D), or Transferrin-Texas Red® (E). A confocal image of an A6 cell that was transiently cotransfected with EGFP-NEDD4L-C2(+) and mCherry-LC3 (autophagosome marker) after a Ca2+ stimulus (F). Blue, green and red channel overlay (A-F). Scale bars are equivalent to 10 μm.

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Additional file 3:

Subcellular localization of EGFP tagged α-ENaC and mCherry-NEDD4L-C2(+) in response to ionomycin treatment in the presence of Ca2+. The movie, taken at 1 frame every 30 seconds, is of an A6 cell that stably expresses α-ENaC-EGFP, was transiently transfected with mCherry-NEDD4L-C2(+), and was incubated in 1.97 mM Ca2+ and 10 μM ionomycin. It is the same A6 cell that is shown in Figure 5, panels A-E.

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Additional file 4:

Subcellular localization of EGFP tagged β-ENaC and mCherry-NEDD4L-C2(+) in response to ionomycin treatment in the presence of Ca2+. The movie, taken at 1 frame every 2 seconds, is of an A6 cell that stably expresses EGFP-β-ENaC, was transiently transfected with mCherry-NEDD4L-C2(+), and was incubated in 1.97 mM Ca2+ and 10 μM ionomycin. It is the same A6 cell that is shown in Figure 5, panels F-J.

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Additional file 5:

Subcellular localization of EGFP tagged β-ENaC and mCherry-NEDD4L-C2(+)-C943A in response to ionomycin treatment in the presence of Ca2+. The movie, taken at 1 frame every 10 seconds, is of an A6 cell that stably expresses EGFP-β-ENaC, was transiently transfected with mCherry-NEDD4L-C2(+)-C943A, and was incubated in 1.97 mM Ca2+ and 10 μM ionomycin. It is the same A6 cell that is shown in Figure 5, panels K-O.

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