1. Volume 116, Issue 3, Pages 610-622 (March 1999)
Transcytosis of immunoglobulin A in the mouse enterocyte occurs through glycolipid raft- and rab17-containing compartments 
Gert H. Hansen, Lise-Lotte Niels-Christiansen, Lissi Immerdal, Walter Hunziker, A.John Kenny, E.Michael Danielsen 
Gastroenterology 
Volume 116, Issue 3, Pages (March 1999)
DOI: /S (99)
Copyright © 1999 American Gastroenterological Association Terms and Conditions

2. Fig. 1
A fraction of IgA is present in low-temperature detergent-insoluble complexes. A total membrane fraction was prepared from mouse small intestinal mucosa (~0.1 g), extracted with CHAPS, and subjected to sucrose density gradient centrifugation as described in Materials and Methods. After centrifugation, the gradient was divided into 11 fractions, and samples of equal volume were analyzed by SDS-PAGE and Western blotting, using primary antibodies to IgA (IgA) or aminopeptidase N (ApN).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Low-temperature insolubility is not acquired during subcellular fractionation. Explants were labeled for 2 hours with 0.5 mCi/mL of [35S]methionine, homogenized in 1 mL of 25 mmol/L HEPES-HCl plus 150 mmol/L NaCl, pH 7.0, and centrifuged at g for 20 minutes to obtain a supernatant of labeled cytosol protein (Cyt•) and a pellet of total membranes. A similar total membrane fraction, prepared in parallel from unlabeled mucosa, was resuspended in the supernatant (containing labeled IgA), incubated on ice for 30 minutes, and pelleted by centrifugation as described above. Both labeled and unlabeled membrane fractions were resuspended in 1 mL of the above buffer, extracted with 1% Triton X-100 for 10 minutes on ice, and centrifuged as described above to obtain a supernatant of low-temperature detergent-soluble protein (S• and S, respectively) and a pellet. The pellet was resuspended, extracted with 1% Triton X-100 for 10 minutes at 37°C, and centrifuged to obtain a supernatant of (solubilized) low-temperature detergent-insoluble protein (I• and I, respectively). IgA was immunopurified from the labeled cytosol and from the labeled and unlabeled membrane fractions as described in Materials and Methods. After SDS-PAGE, IgA was visualized by a PhosphorImager and by Western blotting.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Partial low-temperature detergent insolubility of pIgR/secretory component. Low-temperature detergent-insoluble complexes (DIC) and the corresponding fraction of Triton X-100–solubilized proteins (Sol) were prepared from total membranes of pig small intestinal mucosa by sucrose gradient centrifugation as described previously.26 After SDS-PAGE, samples of the two fractions were analyzed by Western blotting using an antibody to human secretory component (pIgR/SC-blot). After blotting, the gel tracks were stained with Coomassie brilliant blue (protein).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Localization of IgA in mucosal plasma cells. (A) Low-magnification electron micrograph showing an IgA-producing plasma cell. Intense labeling of IgA is seen in the dilated endoplasmic reticulum. (B) Immunogold double-labeling of IgA (13-nm gold particles) and aminopeptidase N (7-nm gold particles). The latter was absent from most of the plasma cells examined, but a single gold particle (arrow) was seen occasionally in the endoplasmic reticulum (ER). (A) Bar = 1 μm; (B) bar = 100 nm.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

6. Fig. 4
Localization of IgA in mucosal plasma cells. (A) Low-magnification electron micrograph showing an IgA-producing plasma cell. Intense labeling of IgA is seen in the dilated endoplasmic reticulum. (B) Immunogold double-labeling of IgA (13-nm gold particles) and aminopeptidase N (7-nm gold particles). The latter was absent from most of the plasma cells examined, but a single gold particle (arrow) was seen occasionally in the endoplasmic reticulum (ER). (A) Bar = 1 μm; (B) bar = 100 nm.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

7. Fig. 5
Localization of IgA in enterocytes. Low-magnification electron micrograph showing the apical part of enterocytes positioned in the upper region of the crypt near the crypt-villus junction. IgA labeling is seen in the apical tubulovesicular compartment (arrowheads), along the lateral plasma membrane, and in lateral endosomes (arrows). Bar = 1 μm.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

8. Fig. 6
Ultrastructural localization of IgA in enterocytes. Immunogold labeling of IgA (13-nm gold particles) in enterocytes. (A and B) Labeling is observed in the basolateral membrane (BM) and in endosomes (EN) positioned along the basolateral plasma membrane. (C) An intense labeling is seen in a tubulovesicular compartment (TVC) positioned in the apical part of the enterocyte. This compartment is particularly prominent in the crypt enterocytes. TJ, tight junction. (D) Labeled vesicles (VE) are frequently observed close to the microvillar membrane (MI). (E) Multivesicular bodies (MB) are frequently observed in the apical part of the enterocytes. IgA labeling in these compartments was usually weak. Bars = 100 nm.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

9. Fig. 7
Colocalization of IgA and aminopeptidase N in enterocytes. Immunogold double-labeling of IgA (13-nm gold particles) and aminopeptidase N (7-nm gold particles) in enterocytes. (A and B) Only a weak labeling of aminopeptidase N (arrowheads) is observed in the basolateral membrane (BM) and in basolateral endosomes (EN) in which IgA labeling is frequent. (C) The labeling of aminopeptidase N is prominent in the apical tubulovesicular compartment (TVC), in which it is colocalized with IgA. (D) Vesicles (VE) labeled for both aminopeptidase N and IgA are frequently observed near the microvillar membrane. (E) Multivesicular bodies (MB) are frequently seen to be significantly labeled for aminopeptidase N, but they are only weakly labeled for IgA. Bars = 100 nm.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

10. Fig. 8
Colocalization of IgA and aminopeptidase N in glycolipid rafts. Immunogold double-labeling of IgA (13-nm gold particles) and aminopeptidase N (7-nm gold particles) of glycolipid rafts prepared from an Mg2+-precipitated membrane fraction as described in Materials and Methods. The micrographs show frequent clustering of IgA and aminopeptidase N on the same vesicular profiles. Bar = 100 nm.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

11. Fig. 9
Specificity of the rab17 antibody. Equal amounts of protein from wild-type (lane 1) or rab17-transfected (lane 2) MDCK cells were fractionated by SDS-PAGE, transferred to nitrocellulose, and probed with the affinity-purified anti-rab17 antibody. A single band of approximately 25 kilodaltons was detected in transfected cells.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

12. Fig. 10
Colocalization of IgA and rab17. Immunogold double-labeling of IgA (13-nm gold particles) and rab17 (7-nm gold particles) in enterocytes. (A and B) Labeling of rab17 (arrowheads) can often be seen along the basolateral membrane (BM) and to a lesser extent in endosomes (EN) positioned along the basolateral plasma membrane. (C) Labeling of rab17 (arrowhead) is weak in the apical part of the enterocyte and is only rarely seen in the apical tubulovesicular compartment (TVC) that is prominently labeled for IgA. MI, microvillar membrane; TJ, tight junction. (D) In vesicles (VE) positioned beneath the microvillar membrane, labeling was observed only for IgA. (E) In multivesicular bodies (MB), weak labeling of IgA was observed, whereas no labeling of rab17 was detected. Bars = 100 nm.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

13. Fig. 11
Newly synthesized IgA is absent from low-temperature detergent-insoluble complexes. Mucosal explants were labeled with 1 mCi/mL [35S]methionine for 15 minutes. After culture, they were homogenized in 1 mL of 25 mmol/L HEPES-HCl plus 150 mmol/L NaCl, pH 7.0. The homogenates were cleared by centrifugation at 500g for 3 minutes, extracted with 20 mmol/L CHAPS on ice for 10 minutes, and subjected to sucrose gradient centrifugation as described in Materials and Methods. After centrifugation, the gradient was divided into 12 fractions, and IgA was immunopurified from each fraction and analyzed by SDS-PAGE as described in Materials and Methods. Newly synthesized (radioactive) IgA was visualized by a PhosphorImager ([35S]Met) and the steady-state amounts of IgA by Western blotting (IgA-blot).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

14. Fig. 12
Low-temperature detergent insolubility of IgA is acquired posttranslationally. Mucosal explants were pulse-labeled for 15 minutes with 0.5 mCi/mL [35S]methionine and chased for the indicated periods. The labeled explant was homogenized and extracted with CHAPS on ice as described in Materials and Methods. The extract was then centrifuged at 20,000g for 20 minutes to obtain a supernatant of low-temperature detergent-soluble proteins (S) and a pellet. The pellet was resuspended and extracted with 20 mmol/L CHAPS at 37°C for 10 minutes. This extract was centrifuged as above to obtain a supernatant of (solubilized) low-temperature detergent-insoluble proteins (I). IgA was immunopurified from both S and I fractions as described in Materials and Methods and subjected to SDS-PAGE. After electrophoresis, the gel was analyzed by a PhosphorImager. (The relatively weak labeling of IgA at 0 minutes of chase was repeatedly observed. We ascribe this to a slow diffusion and uptake of [35S]methionine into the lymphocytes located in the lamina propria of the mucosal explants.)
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

15. Fig. 13
Release of IgA from mucosal explants. (A) Mucosal explants were pulse-labeled for 30 minutes with 1 mCi/mL [35S]methionine at 37°C and chased for 3 hours at either 37°C or 20°C. At the indicated time points, 0.25-mL samples of the media were collected, and the culture dishes replenished with an equal volume of fresh medium. After culture, the explants (Exp) were homogenized in 1 mL of 25 mmol/L HEPES-HCl plus 150 mmol/L NaCl, pH 7.0. The homogenates were solubilized by 20 mmol/L CHAPS for 10 minutes at 37°C and cleared by centrifugation at 20,000g for 20 minutes. IgA was immunopurified from solubilized homogenates and media samples as described in Materials and Methods, subjected to SDS-PAGE, and visualized by a PhosphorImager. (B) A quantitation of the above experiment, expressing the total amount of IgA released into the medium at a given point as a percentage of the amount of IgA remaining in the explant after the 3-hour chase. ▵, 37°C chase; ▴, 20°C chase.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions