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Kwo-Yih Yeh*,‡, Mary Yeh*, Jonathan Glass*, D.Neil Granger‡ 

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Presentation on theme: "Kwo-Yih Yeh*,‡, Mary Yeh*, Jonathan Glass*, D.Neil Granger‡ "— Presentation transcript:

1 Rapid activation of NF-κB and AP-1 and target gene expression in postischemic rat intestine 
Kwo-Yih Yeh*,‡, Mary Yeh*, Jonathan Glass*, D.Neil Granger‡  Gastroenterology  Volume 118, Issue 3, Pages (March 2000) DOI: /S (00) Copyright © 2000 American Gastroenterological Association Terms and Conditions

2 Fig. 1 Activation of intestinal NF-κB after IR. (A) NF-κB activity in nuclear extracts from N-J, internal C-J, and IR-J was determined by EMSA (see Materials and Methods for details). NF-κB activity did not change in the N-J (lane 1) and C-J (lanes 2–5) but was significantly elevated in IR-J from 1 to 12 hours after reperfusion (lanes 6–9). Two undefined complexes (NS) migrated faster than the NF-κB band. Antibodies for p50, but not p65, produced a supershift band (lane 10 vs. lane 11). The presence of 50× DNA competitor diminished all retarded bands (lane 12). Nuclear extracts from isolated epithelial cells (EC) showed that NF-κB activity was low in the C-J and significantly increased in IR-J at 1 hour after reperfusion (lanes 13 and 14). A representative gel of 4 assays using different sets of animal samples. (B) Activation of jejunal NF-κB activity after IR. Radioactivity in EMSA gels as shown in A was quantitated by ImageQuant and statistically analyzed. Data obtained from N-J were designated as the normal level at 0 time point. Data are means ± SE (n = 5 in N-J, 4 in C-J, and 4 in IR-J). Bonferroni test: *P < 0.05 vs. 0 hour. Paired Student t test: *P < 0.05 vs. C-J. Gastroenterology  , DOI: ( /S (00) ) Copyright © 2000 American Gastroenterological Association Terms and Conditions

3 Fig. 1 Activation of intestinal NF-κB after IR. (A) NF-κB activity in nuclear extracts from N-J, internal C-J, and IR-J was determined by EMSA (see Materials and Methods for details). NF-κB activity did not change in the N-J (lane 1) and C-J (lanes 2–5) but was significantly elevated in IR-J from 1 to 12 hours after reperfusion (lanes 6–9). Two undefined complexes (NS) migrated faster than the NF-κB band. Antibodies for p50, but not p65, produced a supershift band (lane 10 vs. lane 11). The presence of 50× DNA competitor diminished all retarded bands (lane 12). Nuclear extracts from isolated epithelial cells (EC) showed that NF-κB activity was low in the C-J and significantly increased in IR-J at 1 hour after reperfusion (lanes 13 and 14). A representative gel of 4 assays using different sets of animal samples. (B) Activation of jejunal NF-κB activity after IR. Radioactivity in EMSA gels as shown in A was quantitated by ImageQuant and statistically analyzed. Data obtained from N-J were designated as the normal level at 0 time point. Data are means ± SE (n = 5 in N-J, 4 in C-J, and 4 in IR-J). Bonferroni test: *P < 0.05 vs. 0 hour. Paired Student t test: *P < 0.05 vs. C-J. Gastroenterology  , DOI: ( /S (00) ) Copyright © 2000 American Gastroenterological Association Terms and Conditions

4 Fig. 2 Western analysis of changes in mucosal p105 and p50 levels and IκB-α degradation and phosphorylation after reperfusion of ischemic intestine. Mucosal cytoplasmic extracts from normal jejunum (N-J, designated as 0 hour) and from IR-J at indicated times were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by Western analysis. (A) p105 and p50 detected with anti p50 antibodies (catalog no. Sc-114; Santa Cruz Biotechnology), which reacted to the nuclear localization signal domain in both p105 and p50 subunits. The decrease of p105 at 0.5 hour after reperfusion was associated with the appearance of p50. (B) Changes in mucosal p105 and p50 levels after reperfusion of ischemic intestine. Western blots as shown in A were quantitated by transmittance densitometry using volume integration and ImageQuant application software. p105 was significantly reduced at 0.5, 1, and 3 hours and returned to the normal level at 6 and 12 hours after reperfusion. p50 appeared at 0.5 hour and decreased to undetectable levels at 3 hours after reperfusion. Bonferroni test: *P < 0.05 vs. 0 hour. (C) IκB-α and phosphorylated IκB-α were detected by anti–IκB-α and phospho-IκB-α antibodies, respectively. Mucosal IκB-α decreased at 1 hour and remained at low levels throughout after reperfusion. Phosphorylated IκB-α showed a 3-fold increase at 0.5 hour, 2- fold at 1 hour, and returned to the basal level at 3 hours after reperfusion. (D) Changes in mucosal IκB-α levels after IR. IκB-α detected in the Western blot was quantitated by transmittance densitometry and analyzed with ImageQuant software. IκB-α decreased significantly at 1 hour and retained significantly lower than the basal level. Bonferroni test: *P < 0.05 vs. 0 hour. Gastroenterology  , DOI: ( /S (00) ) Copyright © 2000 American Gastroenterological Association Terms and Conditions

5 Fig. 2 Western analysis of changes in mucosal p105 and p50 levels and IκB-α degradation and phosphorylation after reperfusion of ischemic intestine. Mucosal cytoplasmic extracts from normal jejunum (N-J, designated as 0 hour) and from IR-J at indicated times were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by Western analysis. (A) p105 and p50 detected with anti p50 antibodies (catalog no. Sc-114; Santa Cruz Biotechnology), which reacted to the nuclear localization signal domain in both p105 and p50 subunits. The decrease of p105 at 0.5 hour after reperfusion was associated with the appearance of p50. (B) Changes in mucosal p105 and p50 levels after reperfusion of ischemic intestine. Western blots as shown in A were quantitated by transmittance densitometry using volume integration and ImageQuant application software. p105 was significantly reduced at 0.5, 1, and 3 hours and returned to the normal level at 6 and 12 hours after reperfusion. p50 appeared at 0.5 hour and decreased to undetectable levels at 3 hours after reperfusion. Bonferroni test: *P < 0.05 vs. 0 hour. (C) IκB-α and phosphorylated IκB-α were detected by anti–IκB-α and phospho-IκB-α antibodies, respectively. Mucosal IκB-α decreased at 1 hour and remained at low levels throughout after reperfusion. Phosphorylated IκB-α showed a 3-fold increase at 0.5 hour, 2- fold at 1 hour, and returned to the basal level at 3 hours after reperfusion. (D) Changes in mucosal IκB-α levels after IR. IκB-α detected in the Western blot was quantitated by transmittance densitometry and analyzed with ImageQuant software. IκB-α decreased significantly at 1 hour and retained significantly lower than the basal level. Bonferroni test: *P < 0.05 vs. 0 hour. Gastroenterology  , DOI: ( /S (00) ) Copyright © 2000 American Gastroenterological Association Terms and Conditions

6 Fig. 2 Western analysis of changes in mucosal p105 and p50 levels and IκB-α degradation and phosphorylation after reperfusion of ischemic intestine. Mucosal cytoplasmic extracts from normal jejunum (N-J, designated as 0 hour) and from IR-J at indicated times were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by Western analysis. (A) p105 and p50 detected with anti p50 antibodies (catalog no. Sc-114; Santa Cruz Biotechnology), which reacted to the nuclear localization signal domain in both p105 and p50 subunits. The decrease of p105 at 0.5 hour after reperfusion was associated with the appearance of p50. (B) Changes in mucosal p105 and p50 levels after reperfusion of ischemic intestine. Western blots as shown in A were quantitated by transmittance densitometry using volume integration and ImageQuant application software. p105 was significantly reduced at 0.5, 1, and 3 hours and returned to the normal level at 6 and 12 hours after reperfusion. p50 appeared at 0.5 hour and decreased to undetectable levels at 3 hours after reperfusion. Bonferroni test: *P < 0.05 vs. 0 hour. (C) IκB-α and phosphorylated IκB-α were detected by anti–IκB-α and phospho-IκB-α antibodies, respectively. Mucosal IκB-α decreased at 1 hour and remained at low levels throughout after reperfusion. Phosphorylated IκB-α showed a 3-fold increase at 0.5 hour, 2- fold at 1 hour, and returned to the basal level at 3 hours after reperfusion. (D) Changes in mucosal IκB-α levels after IR. IκB-α detected in the Western blot was quantitated by transmittance densitometry and analyzed with ImageQuant software. IκB-α decreased significantly at 1 hour and retained significantly lower than the basal level. Bonferroni test: *P < 0.05 vs. 0 hour. Gastroenterology  , DOI: ( /S (00) ) Copyright © 2000 American Gastroenterological Association Terms and Conditions

7 Fig. 2 Western analysis of changes in mucosal p105 and p50 levels and IκB-α degradation and phosphorylation after reperfusion of ischemic intestine. Mucosal cytoplasmic extracts from normal jejunum (N-J, designated as 0 hour) and from IR-J at indicated times were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by Western analysis. (A) p105 and p50 detected with anti p50 antibodies (catalog no. Sc-114; Santa Cruz Biotechnology), which reacted to the nuclear localization signal domain in both p105 and p50 subunits. The decrease of p105 at 0.5 hour after reperfusion was associated with the appearance of p50. (B) Changes in mucosal p105 and p50 levels after reperfusion of ischemic intestine. Western blots as shown in A were quantitated by transmittance densitometry using volume integration and ImageQuant application software. p105 was significantly reduced at 0.5, 1, and 3 hours and returned to the normal level at 6 and 12 hours after reperfusion. p50 appeared at 0.5 hour and decreased to undetectable levels at 3 hours after reperfusion. Bonferroni test: *P < 0.05 vs. 0 hour. (C) IκB-α and phosphorylated IκB-α were detected by anti–IκB-α and phospho-IκB-α antibodies, respectively. Mucosal IκB-α decreased at 1 hour and remained at low levels throughout after reperfusion. Phosphorylated IκB-α showed a 3-fold increase at 0.5 hour, 2- fold at 1 hour, and returned to the basal level at 3 hours after reperfusion. (D) Changes in mucosal IκB-α levels after IR. IκB-α detected in the Western blot was quantitated by transmittance densitometry and analyzed with ImageQuant software. IκB-α decreased significantly at 1 hour and retained significantly lower than the basal level. Bonferroni test: *P < 0.05 vs. 0 hour. Gastroenterology  , DOI: ( /S (00) ) Copyright © 2000 American Gastroenterological Association Terms and Conditions

8 Fig. 3 Changes in jejunal AP-1 binding activity after reperfusion of ischemic intestine determined by EMSA. (A) AP-1 activity, detected as A and B bands, was not altered in C-J and was significantly activated in IR-J throughout the 1–12-hour period of reperfusion. Both A and B bands were diminished in the presence of 50× DNA competitor. A representative gel of 4 assays with different sets of experiments. (B) Supershift assay showed that antibodies against c-fos produced a supershift band in association with the disappearance of A band. Antibodies against either fosB, Fra-1, c-jun, and control antibodies produced neither supershift nor change in binding activity. (C) Changes in AP-1 activity after reperfusion of ischemic intestine. Radioactivity in EMSA gels as shown in 2A was quantitated by ImageQuant and statistically analyzed. AP-1 activity was rapidly elevated 4-fold at 1 hour, remained a 2-fold increase at 3 and 6 hours, and increased to 3-fold higher than the normal level. Data are means ± SE (each data point contains 4 samples). Bonferroni test: *P < 0.05 vs. 0 hour. Paired Student t test: *P < 0.05 vs. C-J. Gastroenterology  , DOI: ( /S (00) ) Copyright © 2000 American Gastroenterological Association Terms and Conditions

9 Fig. 3 Changes in jejunal AP-1 binding activity after reperfusion of ischemic intestine determined by EMSA. (A) AP-1 activity, detected as A and B bands, was not altered in C-J and was significantly activated in IR-J throughout the 1–12-hour period of reperfusion. Both A and B bands were diminished in the presence of 50× DNA competitor. A representative gel of 4 assays with different sets of experiments. (B) Supershift assay showed that antibodies against c-fos produced a supershift band in association with the disappearance of A band. Antibodies against either fosB, Fra-1, c-jun, and control antibodies produced neither supershift nor change in binding activity. (C) Changes in AP-1 activity after reperfusion of ischemic intestine. Radioactivity in EMSA gels as shown in 2A was quantitated by ImageQuant and statistically analyzed. AP-1 activity was rapidly elevated 4-fold at 1 hour, remained a 2-fold increase at 3 and 6 hours, and increased to 3-fold higher than the normal level. Data are means ± SE (each data point contains 4 samples). Bonferroni test: *P < 0.05 vs. 0 hour. Paired Student t test: *P < 0.05 vs. C-J. Gastroenterology  , DOI: ( /S (00) ) Copyright © 2000 American Gastroenterological Association Terms and Conditions

10 Fig. 3 Changes in jejunal AP-1 binding activity after reperfusion of ischemic intestine determined by EMSA. (A) AP-1 activity, detected as A and B bands, was not altered in C-J and was significantly activated in IR-J throughout the 1–12-hour period of reperfusion. Both A and B bands were diminished in the presence of 50× DNA competitor. A representative gel of 4 assays with different sets of experiments. (B) Supershift assay showed that antibodies against c-fos produced a supershift band in association with the disappearance of A band. Antibodies against either fosB, Fra-1, c-jun, and control antibodies produced neither supershift nor change in binding activity. (C) Changes in AP-1 activity after reperfusion of ischemic intestine. Radioactivity in EMSA gels as shown in 2A was quantitated by ImageQuant and statistically analyzed. AP-1 activity was rapidly elevated 4-fold at 1 hour, remained a 2-fold increase at 3 and 6 hours, and increased to 3-fold higher than the normal level. Data are means ± SE (each data point contains 4 samples). Bonferroni test: *P < 0.05 vs. 0 hour. Paired Student t test: *P < 0.05 vs. C-J. Gastroenterology  , DOI: ( /S (00) ) Copyright © 2000 American Gastroenterological Association Terms and Conditions

11 Fig. 4 Northern analysis of jejunal c-fos, neurotensin, and ferritin H mRNA expression after IR. (A) Aliquots of 10 μg RNA isolated from normal (N), control (C), and IR-J (IR) were used for Northern analysis. The same membrane was blotted with each of 32P-labeled c-fos, neurotensin, ferritin H, or 28S ribosomal RNA probes. The figure is a representative of Northern blot of 5 sets of different samples. (B) Changes in jejunal c-fos, neurotensin, and ferritin H mRNA levels after IR. Radioactive signals detected in Northern blots were quantitated with ImageQuant software after normalizing to 28S ribosomal RNA. Data are means ± SE (n = 4 for each sample group). Bonferroni test: †P < 0.05 vs. 0 hour; paired Student t test: *P < 0.05 vs. C-J. Gastroenterology  , DOI: ( /S (00) ) Copyright © 2000 American Gastroenterological Association Terms and Conditions

12 Fig. 4 Northern analysis of jejunal c-fos, neurotensin, and ferritin H mRNA expression after IR. (A) Aliquots of 10 μg RNA isolated from normal (N), control (C), and IR-J (IR) were used for Northern analysis. The same membrane was blotted with each of 32P-labeled c-fos, neurotensin, ferritin H, or 28S ribosomal RNA probes. The figure is a representative of Northern blot of 5 sets of different samples. (B) Changes in jejunal c-fos, neurotensin, and ferritin H mRNA levels after IR. Radioactive signals detected in Northern blots were quantitated with ImageQuant software after normalizing to 28S ribosomal RNA. Data are means ± SE (n = 4 for each sample group). Bonferroni test: †P < 0.05 vs. 0 hour; paired Student t test: *P < 0.05 vs. C-J. Gastroenterology  , DOI: ( /S (00) ) Copyright © 2000 American Gastroenterological Association Terms and Conditions

13 Fig. 5 Pattern of c-fos, neurotensin, and ferritin H expression along the jejunal CV axis detected by Northern blots. (A) NFNAs were isolated from cryostat sections along the CV axis and subjected to Northern blots. To verify the orientation of tissue, expression of cryptdin I was analyzed. A representative Northern blot of 3 assays with different set of samples. (B) Spatial patterns of c-fos, neurotensin, and ferritin H expression along the jejunal CV axis. Radioactive signals from Northern blots as shown in A were quantitated with ImageQuant software after normalizing to 28S ribosomal RNA. Data shown are means ± SE (n = 3 for each sample group). Bonferroni test: *P < 0.05 vs. the immediate-lower level. Gastroenterology  , DOI: ( /S (00) ) Copyright © 2000 American Gastroenterological Association Terms and Conditions

14 Fig. 5 Pattern of c-fos, neurotensin, and ferritin H expression along the jejunal CV axis detected by Northern blots. (A) NFNAs were isolated from cryostat sections along the CV axis and subjected to Northern blots. To verify the orientation of tissue, expression of cryptdin I was analyzed. A representative Northern blot of 3 assays with different set of samples. (B) Spatial patterns of c-fos, neurotensin, and ferritin H expression along the jejunal CV axis. Radioactive signals from Northern blots as shown in A were quantitated with ImageQuant software after normalizing to 28S ribosomal RNA. Data shown are means ± SE (n = 3 for each sample group). Bonferroni test: *P < 0.05 vs. the immediate-lower level. Gastroenterology  , DOI: ( /S (00) ) Copyright © 2000 American Gastroenterological Association Terms and Conditions


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