Protein kinase C–dependent activation of NF-κB in enterocytes is independent of IκB degradation Lori Wilson, Csaba Szabó, Andrew L. Salzman Gastroenterology Volume 117, Issue 1, Pages 106-114 (July 1999) DOI: 10.1016/S0016-5085(99)70556-1 Copyright © 1999 American Gastroenterological Association Terms and Conditions
Fig. 1 (A) Nuclear translocation of NF-κB in immunostimulated DLD-1 cells stimulated by IL-1β and PMA. Electromobility shift assay of NF-κB probe by extracts from DLD-1 cells treated with IL-1β (100 U/mL, upper) and PMA (25 ng/mL, lower) for the indicated times. Unstimulated cells are shown in the lane on the far left. A molar excess of unlabeled NF-κB consensus sequence was added to samples in the far right lane to confirm the specificity of the protein-DNA interaction. (B) Functional activation of NF-κB in immunostimulated DLD-1 cells stimulated by IL-1β and PMA. CT, Control. Luciferase expression in DLD-1 cells transfected with a luciferase construct under the control of three tandem κB sites. Cells were incubated with IL-1β (100 U/mL) or PMA (25 ng/mL) for 8 hours. Luciferase activities were normalized to the corresponding β-galactosidase values to correct for differences in transfection efficiency. Gastroenterology 1999 117, 106-114DOI: (10.1016/S0016-5085(99)70556-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions
Fig. 1 (A) Nuclear translocation of NF-κB in immunostimulated DLD-1 cells stimulated by IL-1β and PMA. Electromobility shift assay of NF-κB probe by extracts from DLD-1 cells treated with IL-1β (100 U/mL, upper) and PMA (25 ng/mL, lower) for the indicated times. Unstimulated cells are shown in the lane on the far left. A molar excess of unlabeled NF-κB consensus sequence was added to samples in the far right lane to confirm the specificity of the protein-DNA interaction. (B) Functional activation of NF-κB in immunostimulated DLD-1 cells stimulated by IL-1β and PMA. CT, Control. Luciferase expression in DLD-1 cells transfected with a luciferase construct under the control of three tandem κB sites. Cells were incubated with IL-1β (100 U/mL) or PMA (25 ng/mL) for 8 hours. Luciferase activities were normalized to the corresponding β-galactosidase values to correct for differences in transfection efficiency. Gastroenterology 1999 117, 106-114DOI: (10.1016/S0016-5085(99)70556-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions
Fig. 2 Nuclear translocation of p50/p65 in DLD-1 cells stimulated by IL-1β and PMA. Electromobility supershift assay of NF-κB probe by extracts from DLD-1 cells treated for 1 hour with IL-1β (100 U/mL, upper) and PMA (25 ng/mL, lower) in lanes B–G. Unstimulated cells are shown in lane A. Samples were incubated with antisera to p50 (lane B), p52 (lane C), p65 (lane D), and c-Rel (lane E). A molar excess of unlabeled NF-κB consensus sequence was added to samples (lane G) to confirm the specificity of the protein-DNA interaction. Gastroenterology 1999 117, 106-114DOI: (10.1016/S0016-5085(99)70556-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions
Fig. 3 IκB degradation in DLD-1 cells stimulated by IL-1β but not by PMA. Western blot analysis of extracts from DLD-1 cells treated with 100 U/mL IL-1β and 25 ng/mL PMA for the indicated times. Unstimulated cells are shown in the leftmost lane. (A) Blots were probed with polyclonal antiserum to IκBα, IκBβ, and IκBγ. (B) Blots are probed with antiserum to the housekeeping protein β-actin as an internal control for loading variation. Gastroenterology 1999 117, 106-114DOI: (10.1016/S0016-5085(99)70556-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions
Fig. 3 IκB degradation in DLD-1 cells stimulated by IL-1β but not by PMA. Western blot analysis of extracts from DLD-1 cells treated with 100 U/mL IL-1β and 25 ng/mL PMA for the indicated times. Unstimulated cells are shown in the leftmost lane. (A) Blots were probed with polyclonal antiserum to IκBα, IκBβ, and IκBγ. (B) Blots are probed with antiserum to the housekeeping protein β-actin as an internal control for loading variation. Gastroenterology 1999 117, 106-114DOI: (10.1016/S0016-5085(99)70556-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions
Fig. 4 Intact IκB does not dissociate from p50/p65 in DLD-1 cells stimulated by PMA. Western blot analysis of extracts from DLD-1 cells treated with 25 ng/mL PMA for the indicated times. Extracts from untreated cells (leftmost lane) and cells treated with 100 μmol/L PMA were immunoprecipitated with antisera to p65 and blots probed with polyclonal antiserum to IκBα and IκBβ. Gastroenterology 1999 117, 106-114DOI: (10.1016/S0016-5085(99)70556-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions
Fig. 5 Nuclear NF-κB is not associated with c-fos in DLD-1 cells stimulated by IL-1β and PMA. Electromobility assay of NF-κB probe by extracts from DLD-1 cells treated for 1 hour with 100 U/mL IL-1β and 25 ng/mL PMA. Extracts from untreated cells are shown at the far left. Supershift studies were performed by incubating samples with antisera to c-fos before electrophoresis. A molar excess of unlabeled NF-κB consensus sequence was added where indicated to confirm the specificity of the protein-DNA interaction. Gastroenterology 1999 117, 106-114DOI: (10.1016/S0016-5085(99)70556-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions
Fig. 6 IκB mRNA in DLD-1 cells stimulated by IL-1β or PMA. Northern blot analysis of extracts from DLD-1 cells treated with 100 U/mL IL-1β and 25 ng/mL PMA for the indicated times. CT, extracts from control experiments (unstimulated cells). Loading variation was controlled for by the level of 18S mRNA. Gastroenterology 1999 117, 106-114DOI: (10.1016/S0016-5085(99)70556-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions
Fig. 7 Stability of IκB in PMA-induced DLD-1 cells is not a result of de novo IκB expression. Western blot analysis of extracts from DLD-1 cells treated with 25 ng/mL PMA for the indicated times. CT, extracts from control experiments (unstimulated cells). Samples were pretreated with (A) actinomycin D (1 μg/mL) or (B) cycloheximide (50 μg/mL) 1 hour before stimulation. In A, CT/Act represents unstimulated cells that were treated with actinomycin D 1 hour before extraction. Blots were probed with polyclonal antisera to IκBα and IκBβ. IκBα appears as the lower single band. IκBβ appears as the slower migrating doublet. Gastroenterology 1999 117, 106-114DOI: (10.1016/S0016-5085(99)70556-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions
Fig. 7 Stability of IκB in PMA-induced DLD-1 cells is not a result of de novo IκB expression. Western blot analysis of extracts from DLD-1 cells treated with 25 ng/mL PMA for the indicated times. CT, extracts from control experiments (unstimulated cells). Samples were pretreated with (A) actinomycin D (1 μg/mL) or (B) cycloheximide (50 μg/mL) 1 hour before stimulation. In A, CT/Act represents unstimulated cells that were treated with actinomycin D 1 hour before extraction. Blots were probed with polyclonal antisera to IκBα and IκBβ. IκBα appears as the lower single band. IκBβ appears as the slower migrating doublet. Gastroenterology 1999 117, 106-114DOI: (10.1016/S0016-5085(99)70556-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions
Fig. 8 Proteasomal inhibition blocks the nuclear translocation of NF-κB in DLD-1 cells stimulated by IL-1β but not by PMA. Electromobility shift assay of NF-κB probe by extracts from DLD-1 cells treated with IL-1β (100 U/mL; lanes B and D) and PMA (25 ng/mL; lanes C, E, and F) for 60 minutes. Unstimulated cells are shown in lane A. Cells were pretreated with the proteasomal inhibitor ALLN (100 μmol/L) before addition of the stimulus (lanes D and E). A molar excess of unlabeled NF-κB consensus sequence was added to samples (lane F) to confirm the specificity of the protein-DNA interaction. Gastroenterology 1999 117, 106-114DOI: (10.1016/S0016-5085(99)70556-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions
Fig. 9 Inhibition of PKC blocks the nuclear translocation of NF-κB in DLD-1 cells stimulated by PMA. Electromobility shift assay of NF-κB probe by extracts from DLD-1 cells treated with IL-1β (100 U/mL; lanes B and D) and PMA (25 ng/mL; lanes C, E, and F) for 60 minutes. Unstimulated cells are shown in lane A. Some cells were pretreated with staurosporine (100 μmol/L) for 1 hour before the addition of the stimulus (lanes C and E). A molar excess of unlabeled NF-κB consensus sequence was added to samples (lane F) to confirm the specificity of the protein-DNA interaction. Gastroenterology 1999 117, 106-114DOI: (10.1016/S0016-5085(99)70556-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions