Fig 1: Expression of sodium/glucose cotransporter 2 (SGLT2)/solute carrier family 5 member 2 antibody (SLC5A2) protein and mRNA in mouse choroid plexus. (A) Immunoblot analysis of the membrane fraction of mouse kidney (K), choroid plexus (CP) and cerebral cortex (Cx) with anti-SGLT2/SLC5A2 antibody. Results from three mice are presented, with samples of different tissues from each mouse arranged in consecutive lanes (indicated by lines above lanes). Results of immunoblotting (A) and densitometry (B) of SGLT2/SLC5A2 protein and RT-qPCR of GAPDH and SLC5A2 mRNA (C) in mouse brains and kidneys. (A) Immunoblot analysis was performed using the membrane fraction of mouse kidney (K), choroid plexus (CP), and cerebral cortex (Cx). Results from three mice are presented, with samples of different tissues from each mouse arranged in consecutive lanes (indicated by lines above lanes). Arrows on the right side of the blot indicate the position of distinct immunoreactive bands at 72, 70, and 26 kDa, respectively. (B) Immunoreactive signal bands with molecular mass around 70 kDa in mouse kidney and choroid plexus are quantified by densitometry. Results from four mice are analyzed and data from a single mouse are presented as a pair of dots connected with a line. (C) Expression status of Slc5a2 and Gapdh mRNA in mouse choroid plexus is compared between choroid plexus and kidne tissues. M, DNA molecular weight marker.
Fig 2: Representative views of SGLT2/SLC5A2 immunohistochemistry in autopsied human brains from all subjects examined. To compare the effect of DM on the immunoreactivity in CPE (CPE) and ependymal cells, results from subjects with or without DM (DM [+] and DM [-], respectively) are presented adjacently. Scale bars: 100 µm.
Fig 3: Immunohistochemical observations of SGLT2/SLC5A2 (A–G) and SGLT1/SLC5A1 (H, I) in human brain (A-G, I) and kidney (H) tissues. (A–D) CPE cells (arrows, A–C) and ependymal cells (arrowheads) (A, B, D) show granular immunoreactivity for SGLT2/SLC5A2 in the cytoplasm. Inset in (B) indicates a magnified image of SGLT2/SLC5A2-positive CPE cells. Some CPE (open arrow) (C) and ependymal cells (open arrowheads) (B, C) are negative for SGLT2/SLC5A2. (E, F) SGLT2/SLC5A2 immunohistochemistry without preabsorption reveals immunoreactivity in the cytoplasm of CPE (arrow) and ependymal (arrowhead) cells (E). Preabsorption of the anti-SGLT2/SLC5A2 antibody with the recombinant antigen abolishes immunoreactivity in CPE (open arrow) and ependymal (open arrowhead) cells (F). (G) The immunoreactivity is undetectable in neurons, glia, and endothelial cells (double open arrows) in the lateral occipitotemporal cortex. (H) Immunohistochemical staining of human kidney samples with the anti-SGLT1/SLC5A1 antibody reveals immunoreactivity on the BBM of the straight part of proximal tubuli. (I) SGLT1/SLC5A1 immunoreactivity is undetectable in CPE (open arrows) and ependymal (open arrowheads) cells. Case 1 (E–G), Case 2 (A), Case 3 (B), Case 4 (C, D, H, I). Scale bars: 100 µm (A, B, G, H, I), 20 µm (inset, B), 50 µm (C–F).
Fig 4: Validation of the specificity of anti-sodium/glucose cotransporter 2 (SGLT2)/solute carrier family 5 member 2 antibody (SLC5A2) antibody used in the present study. (A, B) Immunohistochemical validation of the specificity of an anti-SGLT2/SLC5A2 antibody by immunohistochemical (A-E) and immunoblot (F) analyses using human (A-C) and mouse (D-F) tissues. (A) The human renal cortex containing glomerulus and early proximal convoluted tubules with the BBM is immunoreactive for SGLT2/SLC5A2. (B) The outer stripe containing the straight portion of proximal tubules (asterisks) exhibits no significant immunoreactivity on the BBM. (C) Preabsorption of the antibody with recombinant SGLT2/SLC5A2 protein antigen completely abolishes the immunoreactivity on the BBM of the early proximal tubules in human kidney. (D) In the mouse kidney tissue, in addition to the BBM of early proximal convoluted tubuli, immunoreactivity is observed in the urinary pole of the Bowman's capsule, which is lined with cuboidal epithelial cells (arrows). (E) In the mouse small intestine tissue, where SGLT1/SLC5A1 is known to be expressed, SGLT2/SLC5A2 immunoreactivity is undetectable on the BBM of the columnar absorptive epithelial cells. (F) Immunoblot analysis of the membrane fraction of mouse kidney samples with the anti-SGLT2/SLC5A2 antibody (Antigen -) depicts broad immunoreactive signal bands with molecular masses around 70 kDa, as well as relatively distinct bands with molecular masses of 72 and 70 kDa, and a distinct band with molecular mass of 26 kDa (arrows). Preabsorption of the antibody with recombinant SGLT2/SLC5A2 protein antigen (Antigen +) prevents the immunoreactive signal bands. Scale bars: 50 µm (A-E).
Fig 5: Immunohistochemical observations of SGLT2/SLC5A2 in mouse brains. (A) Granular heterogeneous immunoreactivity is observed in the cytoplasm of CPE (arrows) (A, B) and ependymal (arrowheads) cells. (A') A magnified image shows SGLT2-positive CPE and ependymal cells in the indicated area of (A). (B, C) A part of CPE (open arrow) (B) and ependymal (arrowheads) cells (C) does not show immunoreactivity for SGLT2/SLC5A2. (D) In the cerebral cortex, neurons, glia, and endothelial cells (open double arrows) show very weak or no immunoreactivity for SGLT2/SLC5A2. Scale bars: 50 µm (A-D), 20 µm (A').
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