1. Volume 126, Issue 1, Pages 122-135 (January 2004)
Akt2, phosphatidylinositol 3-kinase, and PTEN are in lipid rafts of intestinal cells: Role in absorption and differentiation 
Xshang Li, Sharon Leu, Alice Cheong, Huiping Zhang, Boris Baibakov, Chris Shih, Morris J. Birnbaum, Mark Donowitz 
Gastroenterology 
Volume 126, Issue 1, Pages (January 2004)
DOI: /j.gastro

2. Figure 1
Akt expression in ileal villus cells. (A) Both Akt1 and Akt2 are expressed in ileal villus cells. BBM, total membranes, and cytosol from rabbit ileal villus cells were prepared as described in the Materials and Methods section. Proteins from each sample (60 μg of BBM or total membrane proteins and 30 μg of cytosol) were separated by 10% SDS-PAGE and analyzed by Western blotting using specific antibodies against Akt1 and Akt2. Multiple arrows indicate the possible differentially phosphorylated Akt2. A representative of 3 similar experiments was shown. All lanes were from the same blot in the same study, with an irrelevant lane removed. (B) Akt1, Akt2, and p-Akt are differentially distributed in native rabbit ileum. Localization of p-Akt (a), Akt1 (b), and Akt2 (c and D) by immunohistochemistry in ileum. d is the enlargement of a portion of C.
Gastroenterology  , DOI: ( /j.gastro )

3. Figure 2
NHE3 and Membrane-associated PI 3-K and Akt2 are colocalized in the apical membrane and endosomes of rabbit ileal villus cells. EGF increases the amounts of Akt2 and p-Akt in both apical and endosomal membranes. Total membranes prepared from ileal villus cells (control and after exposure to EGF 200 ng/mL, 10 minutes, 37°C) were subjected to fractionation using Opti-Prep gradients (10%–30%). Proteins in each fraction were analyzed by SDS-PAGE and Western blotted with antibodies against the proteins indicated. Established membrane markers used include the apical marker sucrase, early endosomal marker EEA1 and Na+/K+-ATPase, a marker of basolateral membranes. (A) Both PI 3-K and Akt2 were mainly localized at the apical membrane of ileal villus cells (lanes 7–10). A portion of PI 3-K and Akt2 was also present at the basolateral membrane (lanes 11–14) and endosomes (lanes 1 and 2). In contrast, more than 50% of Akt1 was present in the endosomal compartment. (B) EGF increased the level of both PI 3-K and active Akt (P-Akt) in both apical and endosomal compartments. In contrast, NHE3 increased in the apical but decreased in the endosomal compartments. As a negative control, sucrase level was not changed by EGF treatment.
Gastroenterology  , DOI: ( /j.gastro )

4. Figure 3
EGF increased the amount of apical membrane NHE3 by a PI 3-K-dependent process. The increased NHE3 occurred only in the apical membrane DRM pool. Ileal segments were incubated either with buffer (cont, lanes 1 and 5), wortmannin (100 nmol/L) for 30 minutes (Wort, lanes 2 and 6), EGF (200 ng/mL) for 10 minutes (EGF, lanes 3 and 7), or with wortmannin (100 nmol/L) for 20 minutes followed by 10 minutes of EGF in the presence of wortmannin (W+E, lanes 4 and 8). BBM isolation and analysis of DS and DRM are as described in the Materials and Methods section. (A) Ponceau S staining for protein as a loading control. (B) The same blot was probed with anti-NHE3 antibody (upper panel), and NHE3 was quantified by ImageQuant (bar graph). Wortmannin inhibited the EGF stimulation of BBM NHE3 under the same conditions that were used in this experiment.10 Wortmannin prevented the EGF-induced increase in the amount of BBM NHE3. AU, arbitrary densitometry units. A representative experiment, which was repeated 10 times, is shown.
Gastroenterology  , DOI: ( /j.gastro )

5. Figure 4
Detergent solubility and raft association of P-Akt2 and PI 3-K in rabbit ileal BBM. (A) Distribution of Akt2 and PI 3-K in the DS and DRM fractions. DS and DRM fractions were prepared from ileal BBM via their solubility in 1% Triton X-100 in MES buffer (pH 6.5). Proteins in the DS and DRM fractions were analyzed by SDS-PAGE and Western blotted with specific antibodies to Akt2 and PI 3-K. Akt2 (upper panel) and PI 3-K (lower panel) were present predominantly in the DRM of ileal BB. Similar results were obtained from 4 independent experiments. (B) Akt2 and PI 3-K are associated with LR. Ileal BBM were solubilized with 1% Triton X-100 at 4°C with or without MβCD pretreatment at 37°C for 30 minutes. Samples were then subjected to Opti-Prep density gradient floatation and fractionated into 11 fractions. Proteins in each fraction were analyzed by SDS-PAGE and Western blotted with different antibodies as indicated. Fractions 1 through 11 were fractions from the highest (35%) (fraction 1) to the lowest density (5%) (fraction 11). Significant amounts of Akt2 and PI 3-K were in the LR fractions that were shifted from lighter fractions (fractions 5 and 6) to heavier fractions (fractions 3 and 4) after MβCD treatment. This was similar to syntaxin 4, a marker for LR of BBM (shifted from fraction 6 to fractions 1–4; A); (B) Double arrows on the left indicate the doublet of Akt2 recognized by Akt antibodies. A representative of 3 similar experiments was shown.
Gastroenterology  , DOI: ( /j.gastro )

6. Figure 5
DS-Associated Akt2 but not DRM- or Raft-associated Akt is active in ileal BBM. (A) Akt2 activities in the DS and DRM pools. In the left panel, Akt2 was immunoprecipitated from DS and DRM fractions of ileal BBM, and activities were measured by in vitro phosphorylation of histone 2B (upper panel). The same blot was probed with anti-Akt2 antibody to show the amount of Akt2 immunoprecipitated (lower panel). In the right panel, 80 μg protein from either DS or DRM fractions of BBM were used for total Akt activity assay using anti-P-Akt. (B) Raft-associated Akt is not active. Anti-P-Akt antibody was used to probe a blot containing fractions collected from a floatation experiment as described in Figure 4B. No active Akt could be detected in the raft fractions (fractions 5 and 6). Akt activity was detected in fractions 1 and 2. Representatives of 2 or 3 experiments are shown.
Gastroenterology  , DOI: ( /j.gastro )

7. Figure 6
EGF stimulates activity of DS-associated Akt2 in ileal BBM but has no effect on the activity of DRM-associated Akt2. Isolated rabbit ileum was incubated with either EGF (200 ng/mL) or buffer (EGF or control) for 10 minutes at 37°C, and BBM were then prepared. DS and DRM fractions were prepared from BBM (as described in Figure 3), and Akt2 was immunoprecipitated. Akt2 activity was analyzed by determining either its phosphorylation of Ser473 using anti-P-Akt (A, upper panel) or its ability to phosphorylate histone 2B (B). Lower panel of panel A shows the amounts of Akt2 immunoprecipitated by stripping the same blot used for the upper panel and reprobing with anti-Akt2 antibody.
Gastroenterology  , DOI: ( /j.gastro )

8. Figure 7
PTEN is predominantly present in the LR fraction of ileal BBM. OptiPrep gradient floatation experiments and sample preparations were done in the presence and absence of MβCD as described in Figure 4B. Blots were probed with anti-PTEN antibody. Arrows indicate wild-type 62-kilodalton PTEN. A representative of 3 experiments is shown.
Gastroenterology  , DOI: ( /j.gastro )

9. Figure 8
Akt expression and membrane distribution in Caco-2 Cells: (A) Akt1 and Akt2 are expressed in Caco-2 cells. Total membranes (60 μg) and cytosol (30 μg) from Caco-2 cells were analyzed by 10% SDS-PAGE and Western blotting with Akt1 and Akt2. A representative of 3 similar studies was shown. (B) Akt2 and PI 3-K are in apical and BLM and early endosomes in Caco-2 cells. Total membranes from Caco-2 cells were subjected to floatation using OptiPrep gradients (10%–30%) as in Figure 2. Similar to the ileal villus cells, PI 3-K and Akt2 were most expressed in the apical membrane with expression also in early endosomes and BLM. Similar results were found in 2 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )

10. Figure 9
Detergent solubility and raft association of Akt2 and PI 3-K in Caco-2 membranes. (A) Distribution of Akt2 and PI 3-K in DS and DRM of Caco-2 membranes. DS and DRM fractions of total Caco-2 membranes were prepared and Western blotted as in Figure 4A. Akt2 (upper panel) was equally distributed in DS and DRM and PI 3-K (lower panel) was mostly in the DRM. Similar results were obtained in 3 experiments. (B) Akt2 and PI 3-K are in LR in Caco-2 membranes. Caco-2 membranes were solubilized in 1% Triton X-100 at 4°C with and without MβCD pretreatment, subjected to OptiPrep floatation, and processed as in Figure 4B. Syntaxin 3 was used as a marker for LR. Significant amounts of Akt2 and PI 3-K behaved similarly to syntaxin 3, shifting from fractions 4 and 5 to 1 and 3 with MβCD treatment.
Gastroenterology  , DOI: ( /j.gastro )

11. Figure 10
EGF treatment stimulates Caco-2 Akt2 activity, and Akt2 is active in both DS and DRM/LR pools of Caco-2 cells. Filter-grown Caco-2 cells were incubated with EGF (200 ng/mL) for 10 minutes. Akt2 in total membrane, DS/DRM pools, or lipid rafts of membranes were immunoprecipitated and analyzed for Akt2 amount and activity (P-Akt) as described in Figures 5 and 6. (A) EGF increased the levels of both Akt2 and P-Akt2 in total membranes. (B) Akt2 in both DS and DRM pools is active. DS/DRM were prepared from total membranes and then subjected to IP of Akt2, followed by activity determination with histone 2B as substrate. (C) Akt2 in both LR and non-LR fractions was active. Gradient floatation of total membranes was done as described in Figure 5B. Akt2 was IP from the first 5 fractions of the gradient. Similar results were obtained from 2 or 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )

12. Figure 11
PTEN is not in LR in Caco-2 (A) and HT-29 (B) cell membranes. OptiPrep gradients were carried out for membrane localization and performed on total membranes of Caco-2 and HT-29 cells as in Figure 4A, with blots probed with anti-PTEN antibody. Large arrows indicate wild-type 62-kilodalton PTEN, and small arrows indicate ∼48-kilodalton bands also recognized by the same antibody. A single experiment representative of 3 experiments is shown.
Gastroenterology  , DOI: ( /j.gastro )

13. Figure 12
Amount and activity of Akt2 were elevated in differentiated Caco-2 and HT29-18 cells compared with those from undifferentiated cells, and a portion of elevated Akt2 was partitioned into LR. Caco-2 cells were grown on filters to ∼50% confluency (undifferentiated cells) or 7 days postconfluency (7DPC, differentiated cells). HT29-18 cells were grown on plastic petri dishes to 7 days postconfluency in medium containing either glucose (Glu, undifferentiated cells) or galactose (Gal, differentiated cells). Total membranes were prepared. Akt2 was IP, and levels of Akt2 and P-Akt2 were analyzed by SDS-PAGE and Western blotting. (A) Levels of both Akt2 and P-Akt2 were significantly elevated in differentiated Caco-2 (A, part a) and HT29-18 (A, part b) cells. To show that an equal amount of proteins were used for IP of Akt2, the profiles of total membrane proteins used for IP were transferred onto nitrocellulose membranes and visualized by Ponceau S staining (35 μg protein each lane). (B) The levels of both PI 3-K and Akt2 were elevated in LR of differentiated HT29-18 cells (+Gal) compared with the undifferentiated cells (+Glu). OptiPrep gradient floatation experiments and sample preparations were done as described in Figure 4B.
Gastroenterology  , DOI: ( /j.gastro )