1. Volume 133, Issue 1, Pages 232-243 (July 2007)
p27kip1 Regulates cdk2 Activity in the Proliferating Zone of the Mouse Intestinal Epithelium: Potential Role in Neoplasia 
Helena J.M. Smartt, Sandra Guilmeau, Shannon V. Nasser, Courtney Nicholas, Laura Bancroft, Sharon A. Simpson, Nancy Yeh, Wancai Yang, John M. Mariadason, Andrew Koff, Leonard H. Augenlicht 
Gastroenterology 
Volume 133, Issue 1, Pages (July 2007)
DOI: /j.gastro
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2. Figure 1
Protein expression and complex formation among and activity of cell cycle regulators along the small intestinal crypt-villus axis. Five to 50μg total cellular protein extracts from fractions 1–10 were analyzed by Western blotting for expression of markers of intestinal epithelial cell proliferation (PCNA) or of villus differentiation (villin) or α-tubulin (to assess protein loading) (A) and for cell cycle regulators: cyclin D1 and D2, cyclin E, cyclin A (B); cdk2, cdk4 (C); and p57, p21, p16, and p27 (D). Results are representative of at least 3 independent experiments (5 for p27 immunoblotting). To analyze the extent of interaction between G1/S-regulatory cyclins and cdks, 1000 μg total cellular protein extracts from fractions 1–10 were immunoprecipitated using cdk4 (E) or cyclin A (F) antibodies and immunoblotted with cyclin D1/D2 or cdk2 antibodies (E and F, top panels). Fractions 1 and 2 were combined because of insufficient protein yields of fraction 1. Cdk expression in input lysates is shown (E and F, middle panels), and α-tubulin levels confirm equal loading (E and F, bottom panels). To analyze the cdk activity of cdk4 and cdk2-containing complexes, 150 μg total cellular protein extracts from fractions 1–10 were immunoprecipitated using a control rabbit anti-mouse immunoglobulin and a polyclonal cyclin D1, D2, and D3 antibody mixture (G) or a polyclonal anti-cdk2 antibody (H). Kinase activity was determined by comparing the extent of incorporation of γ-32P-ATP into recombinant (Rb or histone H1) substrate proteins by cyclin or cdk immunoprecipitates (G and H, second panels) relative to those using the control antibody (G and H, upper panels). For each kinase assay, cdk expression in input lysates is shown (G and H, third panels), and α-tubulin levels confirm equal protein loading (G and H, lower panels).
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Confirming maximal p27 expression in small intestinal crypt fractions. (A) Five hundred micrograms total cellular protein extracts from fractions 1–10 were immunoprecipitated using a polyclonal p27 antibody and analyzed by immunoblotting with a second monoclonal p27 antibody to confirm the identity of the p27 band seen in Figure 1D. Fractions 1 and 2 were combined because of insufficient protein yields of fraction 1. (B) Fifty micrograms total cellular protein extracts from fractions 1–10 from wild-type (“wt”) and p27Δ51/Δ51 (“Δ51”) mice were analyzed for p27 expression by immunoblotting.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
p27, PCNA, CD3, and lysozyme expression in normal duodenal crypt epithelium. Sections of wild-type (A; C–H) and p27Δ51/Δ51 (B) murine duodenum were examined for expression of p27 (A, B, D, and H), PCNA (C), the T-cell marker CD3 (E, F, and H), and the Paneth cell marker lysozyme (G) by immunohistochemistry. H shows double staining for p27 (light pink) and CD3 (dark crimson/black): solid arrows in A and H indicate strongly p27-positive nuclei; solid arrowheads in E and H indicate intraepithelial lymphocytes; white asterisks in H indicate lamina propria T cells in the core of 3 adjacent villi (only the lower villus is shown). Brackets in D, E, and H indicate crypt regions. Scale bar, 20 μm.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

5. Figure 4
p27 interacts with cdk4 and cdk2 mainly in crypt cell fractions from small intestine. (A) One thousand micrograms total protein extracts from fraction 10 cells were immunoprecipitated using either polyclonal cdk4, cdk2, or control rabbit antibodies and analyzed by immunoblotting with monoclonal p27 (top panel) or p21 (bottom panel) antibodies. (B) To analyze p27 and cdk4 (top panel) or cdk2 (middle panel) interactions along the crypt-villus axis, 1000 μg total cellular protein extracts from fractions 1–10 were immunoprecipitated using polyclonal cdk4 (top panel) or cdk2 (middle panel) antibodies and analyzed by immunoblotting with an p27 antibody. Input lysates were also analyzed by α-tubulin immunoblotting for equal protein loading (bottom panel). Fractions 1 and 2 were combined because of insufficient protein yields of fraction 1. Immunoprecipitation-immunoblotting and analyses in C were carried out as in A but using 300 μg total protein extracts from the rapidly proliferating colon cancer cell line HCT-8.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
Levels of cyclin D-cdk4 complexes in wild-type and p27Δ51/Δ51 small intestinal epithelial crypt cells. (A) One thousand micrograms total cellular protein extracts from crypt intestinal epithelial cells from wild-type (“wt”) and p27Δ51/Δ51 (“Δ51”) mice were immunoprecipitated using anti-cdk4 and analyzed by immunoblotting with an antibody recognizing cyclin D1 and D2 to assess the extent of cyclin-cdk complex formation. (B) Input lysates were analyzed by immunoblotting for cdk4 (top panel), cyclin D1/2 (middle panel), and α-tubulin (bottom panel).
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Cyclin D-associated and cdk2 kinase activities and PCNA-positive cell counts in wild-type and p27Δ51/Δ51 small intestinal epithelial crypt cells. One hundred to 400 μg total protein extracts from wild-type (“wt”) or p27Δ51/Δ51 (“Δ51”) fraction 10 cells were immunoprecipitated with control rabbit anti-mouse antibody and an cdk2 antibody (A) or an cyclin D1, D2, and D3 mixture (B). Immunoprecipitates were then assayed for kinase activity toward histone H1 (A, top panel) or Rb (A, second panel; B) substrates in the presence (A; B: top panels) or absence (A; B: second panels) of γ-32P-ATP, and reactions were subjected to SDS-PAGE. Kinase activity was assessed by autoradiography of dried electrophoretic gels (A; B: top panels) or by immunoblotting using phospho-specific antibodies against the preferential cdk2 target site Rb (ser807/11) (A, second panel) or the preferential cdk4/6 target site Rb (ser780) (B, second panel). Bar charts (A; B: lower panels) show background-adjusted densitometric data (presented as mean cdk2 [A] or cyclin D-associated [B] kinase activity + 1 SD) from 3 independent repeat experiments. An asterisk indicates a significant difference (P < .05) between kinase activity in wild-type and p27Δ51/Δ51 samples. The levels of cdk4 and cdk2 proteins in kinase assay input lysates were assessed by immunoblotting, and equal protein loading was confirmed by α-tubulin expression levels (C). (D) PCNA-positive cell counts in wild-type and p27Δ51/Δ51 duodenal crypts (presented as mean number of positive nuclei per crypt + 1 SD, n = 4; **indicates a highly significant difference, P < .01).
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Cdk2 kinase activity in wild-type and p27Δ51/Δ51 large intestinal epithelial crypt cells. One hundred micrograms total protein extracts from wild-type (“wt”) or p27Δ51/Δ51 (“Δ51”) large intestinal crypt cells were immunoprecipitated with control rabbit anti-mouse or an cdk2 antibody and immunoprecipitates assayed for kinase activity toward histone H1 (A, top panel). The bar chart (A, lower panel) shows background-adjusted densitometric data (presented as mean cdk2 kinase activity + 1 SD) from 3 independent repeat experiments. An asterisk indicates a significant difference (P < .05) between kinase activity in wild-type and p27Δ51/Δ51 samples. The level of cdk2 protein in kinase assay input lysates was assessed by immunoblotting and equal protein loading confirmed by α-tubulin expression levels (B).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

9. Figure 8
Cyclin D-associated and cdk2 kinase activities in wild-type and p21−/− small intestinal epithelial crypt cells. (A and B) One hundred fifty micrograms total cellular protein extracts from wild-type (“wt”) or p21−/− (“−/−”) fraction 10 cells were immunoprecipitated with control rabbit anti-mouse antibody and an cdk2 antibody (A) or an cyclin D1, D2, and D3 mixture (B). Immunoprecipitates were then assayed for kinase activity toward histone H1 (A) or Rb (B) substrate in the presence of γ-32P-ATP and reactions subjected to SDS-PAGE. Kinase activity was assessed by autoradiography of dried electrophoretic gels. Results are representative of 2 (A) or 3 (B) independent repeat experiments. The levels of cdk2 and cdk4 proteins in kinase assay input lysates were assessed by immunoblotting, and equal protein loading was confirmed by α-tubulin expression levels (C).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions