Volume 7 issue 11 November 2006
Defective intracellular trafficking of uromodulin mutant isoforms
Ilenia Bernascone, Stefano Vavassori, Alessio Di Pentima, Sara Santambrogio, Giuseppe Lamorte, Antonio Amoroso, Francesco Scolari, Gian Marco Ghiggeri, Giorgio Casari, Roman Polishchuk, Luca Rampoldi

Supplementary information

Supplementary Figure S1: (a) Western Blot analysis of uromodulin in the soluble (L) and unsoluble (P) fractions of RIPA buffer extracts from HEK293 cells transiently transfected with the EGFP encoding plasmid pcDNA3x(+)MyEGFP and either wild type or mutant uromodulin. Cell were lysed 14h after transfection. While the majority of uromodulin is detected as a mature form in the unsoluble fraction for the wild type, mutants are enriched in the immature form, mainly in the soluble fraction.Alpha-t ubulin is shown as a loading control. (b) Western Blot analysis of EGFP as a control of transfection efficiency. Equal amounts of soluble fractions from (a) were loaded. Alpha-tubulin is shown as a loading control

Figure S1 (.tif)

Supplementary Figure S2: (a,b) Immunofluorescence analysis showing intracellular distribution of different uromodulin mutant isoforms in transiently transfected HEK293 cells. Cells were transfected, methanol-fixed 6h after transfection and incubated with anti-uromodulin and anti-giantin (Golgi marker). Bar = 35µm

Figure S2a (.tif)
Figure S2b (.tif)

Supplementary Figure S3: (a,b) Immunofluorescence analysis showing intracellular distribution of different uromodulin mutant isoforms in transiently transfected HEK293 cells. Cells were transfected, methanol-fixed 6h after transfection and incubated with anti-uromodulin and anti-calreticulin (ER marker). Bar = 35µm.

Figure S3a (.tif)
Figure S3b (.tif)

Supplementary Figure S4: Immunofluorescence analysis showing plasma membrane distribution of wild type and mutant uromodulin in transiently transfected HEK293 cells. Cells were transfected, PFA-fixed 6h after transfection (unpermeabilised cells) and incubated with anti-uromodulin and lectin-TRITC that was used to stain the plasma membrane. Bar = 35µm

Figure S4 (.tif)

Supplementary Movie S1: Time-lapse video of MDCK cells expressing wild type uromodulin_EGFP. Confocal digital images were collected at 15 s intervals for a duration of 20 min after selective photobleaching in the Golgi apparatus region

Movie S1 (.mov)

Supplementary Movie S2: Time-lapse video of MDCK cells expressing mutant (C150S) uromodulin_EGFP. Confocal digital images were collected at 15 s intervals for a duration of 20 min after selective photobleaching in the Golgi apparatus region.

Movie S2 (.mov)

Supplementary Movie S3:Time-lapse video of MDCK cells expressing mutant (C347G) uromodulin_EGFP. Confocal digital images were collected at 15 s intervals for a duration of 20 min after selective photobleaching in the Golgi apparatus region.

Movie S3 (.mov)

Supplementary Movie S4: Time-lapse video of MDCK cells expressing wild type uromodulin_EGFP. Confocal digital images were collected at 15 s intervals for a duration of 25 min after selective photobleaching in the plasma membrane region

Movie S4 (.mov)

Supplementary Movie S5: Time-lapse video of MDCK cells expressing C150S uromodulin_EGFP. Confocal digital images were collected at 15 s intervals for a duration of 25 min after selective photobleaching in the plasma membrane region

Movie S5 (.mov)

Supplementary Movie S6: Time-lapse video of MDCK cells expressing C347G uromodulin_EGFP. Confocal digital images were collected at 15 s intervals for a duration of 25 min after selective photobleaching in the plasma membrane region

Movie S6 (.mov)