Figure 1.
Amino acids sequences of SLC2A9 N-termini of 2 wild types and mutant constructs.
A. Two isoforms, SLC2A9-S and SLC2A9-L, have different N-termini of 21 and 50 amino acids, respectively. B, C. Deleted or mutated constructs of SLC2A9-S (B) and -L (C) were shown.
Figure 2.
Characterization of isoform specific SLC2A9 antibodies.
30 µg of COS cell lysate transfected with mock or SLC2A9s were separated by SDS-PAGE and Western blotting was performed with anti-SLC2A9-S (A), antigen pre-absorbed anti- SLC2A9-S (B), anti-SLC2A9-L (C), or antigen pre-absorbed anti-SLC2A9-L (D) antibodies. COS cells were transfected with GFP tagged SLC2A9-S (E and K) or SLC2A9-L (H and N) and immunofluorescence was performed with anti-SLC2A9-S (F and I) or anti-SLC2A9-L (L and O) antibodies. Overlay images were shown in G, J, M and P. The scale bar of 40 µm was shown in K.
Figure 3.
SLC2A9 expression in human kidney.
SLC2A9-S (A,D) or SLC2A9-L (G,J) was stained together with fluorescein-Lotus Tetragonolobus lectin (LTL; green) (B,H) or rhodamine - Dolichos Biflorus agglutinin (DBA; red) (E,K). Overlay images staining with nucleus (blue) and with phase contrast images were shown in C, F, I and L. The scale bar of 30 µm was shown in L.
Figure 4.
Expression of fluorescent-tagged SLC2A9 isoforms in MDCK cells.
GFP-tagged (green) SLC2A9 -S (A) or -L (B) constructs were transfected into MDCK cells. Cell surface of the apical or basolateral membrane was biotinylated and visualized in red. Confocal images of xy and z axes were obtained and the white scale bar was 30 µm.
Figure 5.
Localization of N-terminal deletion constructs of SLC2A9-S.
GFP-tagged Short_16AAdel construct (green), which was deleted with N-terminal 16 amino acids from SLC2A9-S, was transfected into MDCK cells. Cell surface of the apical or basolateral membrane was biotinylated and visualized in red. Conforcal images of xy and z axes were obtained and the magnification is the same as Fig. 3.
Figure 6.
Significance of the putative di-leucine motif in SLC2A9-L on basolateral sorting.
A. GFP-tagged Long_33-34AA (green), in which 33rd and 34th leucine-leucine was changed to alanines were expressed in MDCK cells. B. GFP-tagged Long_32-36del (green), in which 32nd–36th amino acids including the putative di-leucine motif was deleted, were expressed in MDCK cells. Cell surface of the apical or basolateral membrane was biotinylated and visualized in red. Confocal images of xy and z axes were obtained and the magnification is the same as Fig. 3.
Figure 7.
Effect of N-terminal deletion or exchange of SLC2A9-L.
Long_25AAdel (A) or Long_30AAdel (B) constructs, in which N-terminal 25 or 30 amino acids were deleted from SLC2A9-L, respectively, Long_27-34A (C), in which amino acids from 27th and 31st were changed to alanines, or Long_27-31del (D), in which 5 amino acids from 27th and 31st were deleted, were transfected into MDCK cells. These GFP-tagged constructs were visualized in green. Cell surface of the apical or basolateral membrane was biotinylated and visualized in red. Confocal images of xy and z axes were obtained and the magnification is the same as Fig. 3.
Figure 8.
Effect of N-terminal deletion of SLC2A9-L near the transmembrane side.
GFP-tagged Long_30-50del (A), in which amino acids from 30th to 50th were deleted, or GFP-tagged Long_25-50del (B), in which amino acids from 25th to 50th were deleted from SLC2A9-L were expressed in MDCK cells. These constructs were visualized in green. Cell surface of the apical or basolateral membrane was biotinylated and visualized in red. Conforcal images of xy and z axes were obtained and the magnification is the same as Fig. 3.
Figure 9.
Intracellular localization of SLC2A9 deletions.
GFP-tagged UARTv1-L wild-type (A), Long_25AAdel (B) and Long_30AAdel (C) were expressed in MDCK cells. These constructs were visualized in green. Lysosome of the MDCK cell was stained with lysotracker in red. Confocal images of xy obtained and the white scale bar was 20 µm.
Figure 10.
Comparison of interspecies amino acid sequences around putative LL motif in various transporters.
LL in SLC2A9 was not conserved across species. LL in NKCC1, ENT2, sat-1, GLUT8 (SLC2A8) has been shown to be important in trafficking of these transporters (see text) and amino acid sequences surrounding LL were conserved.