1. Volume 61, Issue 4, Pages 1351-1364 (April 2002)
NHE3 and NHERF are targeted to the basolateral membrane in proximal tubules of colchicine-treated rats 
Ivan Sabolić, Carol Mirna Herak-Kramberger, Marija Ljubojević, Daniel Biemesderfer, Dennis Brown 
Kidney International 
Volume 61, Issue 4, Pages (April 2002)
DOI: /j x
Copyright © 2002 International Society of Nephrology Terms and Conditions

2. Figure 1
Immunostaining of microtubules in cells of the convoluted (A, B) and straight (C, D) proximal tubule of control (A, C) and colchicine-treated (B, D) rats. In control animals (A, C), microtubules were mainly arranged in bundles along the apical/basal axis of proximal tubule cells. Staining was more intense in the subapical region, reflecting the reticular network of microtubules and other microfilaments in the terminal web. In colchicine-treated rats (B, D), microtubules were largely lost in the cells of both proximal tubule segments, but some residual staining was observed in the subapical region (bar = 40 μm).
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions

3. Figure 2
Immunostaining of NHE3 in cells of the convoluted (A, B) and straight (C, D) proximal tubule of control (A, C) and colchicine-treated (B, D) rats. In convoluted proximal tubules of control animals (A), NHE3 was present in the BBM (arrow) and subapical endosomes (arrowhead), whereas in the cells of straight proximal tubule (C), the antigen was present in the BBM (arrow) and in numerous, randomly scattered cytoplasmic organelles (arrowhead). Following colchicine treatment, staining of the BBM was diminished in the proximal convoluted tubule (B), the antigen was detected intracellularly, and the basolateral domain was also stained in many cells (arrowheads). In the cells of straight proximal tubule (D), the BBM staining was unaffected by colchicine treatment, a granular intracellular staining was rarely visible, and the basolateral membrane was clearly stained (arrowheads; bar = 20 μm).
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions

4. Figure 3
Average pixel intensity of NHE3 staining in the brush-border (A; BBM) and basolateral membrane (B; BLM) of cells of convoluted and straight proximal tubule segments from control and colchicine-treated rats. Values are means ± SEM of the number of tissue samples (one sample = tissue sections from one rat) indicated by (N). In colchicine-treated rats, the average pixel intensity of the BBM staining was strongly decreased in the cells of convoluted but not of straight proximal tubule, whereas the pixel intensity of the basolateral staining was significantly increased in the cells of both proximal tubule segments. * vs. control, P <
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions

5. Figure 4
Western blot of NHE3 and 31 kD H+-ATPase subunit in BBM isolated from the kidney cortex (A) and outer stripe (B) of control (lanes 1-4) and colchicine-treated (lanes 5-8) rats. Following colchicine-treatment, the abundance of NHE3 was greatly decreased in the cortical but not outer stripe BBM, whereas the 31 kD protein was diminished in membrane preparations from both tissue zones. The densitometric evaluation of these bands is shown in Table 1.
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions

6. Figure 5
Immunogold labeling of NHE3 in the apical (A, C) and basolateral (B, D) domains of epithelial cells from the S1 segment of control (A, B) and colchicine-treated (C, D) rats. In control animals, heavy labeling at the external side of the BBM (arrow) and the internal side of subapical vesicles (arrowhead) (A) was found. There was no labeling of the basolateral membrane (B). In colchicine-treated rats, the BBM was shorter in height and irregular, and gold particles were less numerous over the membrane (C). The basolateral membrane (D, arrow) and intracellular vesicles (D, arrowhead) were clearly labeled in these cells from colchicine-treated rats (bar = 0.5 μm).
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions

7. Figure 6
Immunogold labeling of NHE3 in the apical (A, D) and basolateral (C, E) domains of the S3 proximal tubule cells from control (A,B, C) and colchicine-treated (D, E) rats. In control animals, heavy labeling was present at the external side of the BBM (A, arrow). Some label was also found over subapical tubulovesicles (A, arrowhead) and over the membrane of lysosome-like organelles inside the cell (B, arrow). The basolateral membrane was not labeled (C). In colchicine-treated rats, the BBM remained strongly labeled (D), labeled intracellular organelles were rare (E, arrowhead), whereas labeling of the external side of the basolateral membrane was prominent (E, arrow; bar = 0.5 μm).
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions

8. Figure 7
Localization of NHE3 (green fluorescence) and NHERF (red fluorescence) and their colocalization (yellow fluorescence) in proximal tubules of control (A-C, G-I) and colchicine-treated (D-F, J-L) rats. In the S1/S2 segments of control rats (A-C), NHE3 (A) and NHERF (B) were restricted to the apical cell domain in the proximal (PT) and distal tubules (DT), where they colocalized (C); no significant staining was observed in the basolateral membrane. In the same segments of colchicine-treated rats, both NHE3 (D) and NHERF (E) were present on the basolateral surface (arrowheads) in addition to the apical membrane, and exhibited a clear colocalization (F). In the S3 segment of control animals (G-I), NHE3 (G) and NHERF (H) were abundant in the apical cell domain, where they completely colocalized (I), but were negative in the basolateral membrane. In this preparation, NHE3-positive intracellular organelles were rare. In colchicine-treated rats (J-L), however, NHE3 (J) and NHERF (K) staining in the proximal tubule S3 segments (PT) was still strong in the apical cell domain, but was also clearly detectable in the basolateral membrane domain (arrowheads), where they were colocalized (L). However, many NHE-positive intracellular organelles within the S3 proximal tubule cells were negative for NHERF (bar = 25 μm).
Kidney International  , DOI: ( /j x)
Copyright © 2002 International Society of Nephrology Terms and Conditions