spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Espinosa, L.
Right arrow Articles by Bigas, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Espinosa, L.
Right arrow Articles by Bigas, A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

p65-NF{kappa}B synergizes with Notch to activate transcription by triggering cytoplasmic translocation of the nuclear receptor corepressor N-CoR

Lluís Espinosa, Sara Santos, Julia Inglés-Esteve, Pura Muñoz-Canoves and Anna Bigas*

Centre Oncologia Molecular, Institut de Recerca Oncologica, Hospitalet, Barcelona 08907, Spain



View larger version (16K):

[in a new window]
  		Fig. 1. Synergistic effect of p65-NF{kappa}B and Notch-IC in the transactivation of the Hes1 promoter. (A) N1-IC (0.5 µg) was cotransfected along with the Hes1-luc reporter (0.75 µg) in NIH-3T3 cells and increasing amounts of p65 wt. Lower panels show N1-IC and p65 protein levels. Protein loading for each lane was normalized according to ß-galactosidase expression. (B,C) p65-NF{kappa}B can synergize with different Notch-IC homologs. 0.5 µg of N2-IC (B) or N3-IC (C) were cotransfected with p65 wt (0.5 µg) along with the Hes1-luc reporter plasmid (1µg) in NIH-3T3 cells. (D) Synergistic effect of p65-NF{kappa}B and Notch-IC is not modified by ectopic expression of p50-NF{kappa}B. NIH-3T3 cells were cotransfected with different combinations of equivalent amounts (0.5 µg) of p50 subunit, N1-IC and p65 wt expression vectors. Luciferase activity is represented as the fold induction relative to the basal level measured in cells transfected with empty vector. The average and standard deviation of duplicates from a representative experiment are presented.

 


View larger version (24K):

[in a new window]
  		Fig. 2. Transcriptional activation of NF{kappa}B-dependent genes is not required for p65-mediated upregulation of the Hes1 promoter. NIH-3T3 cells were transfected with increasing amounts of I{kappa}B{alpha}32-36 in the presence of N1-IC (0.5 µg) and p65 wt (0.5 µg). The reporter constructs were (A) 2x{kappa}B-IL2-luc (1 µg) or (B) Hes 1-luc (1 µg). (C) Immunofluorescence assays to determine the subcellular localization of N1-IC and p65wt when coexpressed with I{kappa}B{alpha}32-36 in NIH-3T3 cells. Cells were cotransfected with N1-IC, p65 wt and I{kappa}B{alpha}32-36. N1-IC was detected with the 9E10 antibody and a FITC-labeled secondary antibody. p65 was detected with {alpha}-p65 and a Cy3-conjugated secondary antibody. (D) Luciferase fold activation obtained by cotransfecting the indicated amounts p65{Delta}TA with N1-IC (0.5 µg) and the Hes1-luc reporter (1 µg). Luciferase activity is presented as the fold induction relative to the basal level measured in cells transfected with empty vector. The average and standard deviation of duplicates from a representative experiment are presented. Lower panels show N1-IC and p65{Delta}TA protein levels. Protein loading for each lane was normalized according to ß-galactosidase expression.

 


View larger version (12K):

[in a new window]
  		Fig. 3. The Hes1 promoter is repressed by N-CoR, and this effect is reversed by p65-NF{kappa}B overexpression. (A) N-CoR represses N1-IC activation of the Hes1 promoter. Indicated amounts of N-CoR were cotransfected with N1-IC expression vector (0.5 µg) into NIH-3T3 cells. (B) p65{Delta}TA can revert inhibition mediated by N-CoR. Increasing amounts of p65{Delta}TA were cotransfected with N1-IC (0.5 µg) and N-CoR (2 µg). Luciferase activity is presented as the fold induction relative to the basal level measured in cells transfected with empty vector. The average and standard deviation of duplicates from a representative experiment are presented. (C) Hes1 expression in 293T cells transfected with N2-IC and N-CoR expression vectors or coexpressed with p65wt is shown in the upper panel. Expression of N2-IC plasmid to compare transfection efficiency is shown in the lower panel.

 


View larger version (36K):

[in a new window]
  		Fig. 4. N-CoR is translocated to the cytoplasm in the presence of p65{Delta}TA. (A) Immunofluorescence staining of 293T cells transfected with N-CoR in the presence (lower panels) or absence (upper panels) of p65{Delta}TA. N-CoR-flag was detected by incubating with {alpha}-flag antibody and a FITC-conjugated secondary antibody (left panels), {alpha}-p65 staining is represented in the middle panels corresponding to Cy3 signal and nuclei were visualized by DAPI staining (right panels). Ectopic expression of p65{Delta}TA does not affect N-CoR protein levels. (B) Immunoblotting analysis of whole cell extracts isolated from 239T cells transfected with N1-IC and N-CoR-flag (lane 1) and N1-IC, N-CoR-flag and p65{Delta}TA (lane 2). Cell lysates were separated on a 6% acrylamide gel, and immunoblots were performed with the following antibodies: {alpha}-flag (N-CoR), {alpha}-N1(N1-IC) and {alpha}-p65. Protein loading for each lane was normalized according to ß-galactosidase expression and confirmed with {alpha}-N1 immunoblot. The western blot for p65 shows endogenous p65 (upper band) and transfected p65{Delta}TA (lower band).

 


View larger version (32K):

[in a new window]
  		Fig. 5. p65-NF{kappa}B nuclear export is required for cytoplasmic retention of N-CoR. (A) Immunofluorescence staining of 293T cells transfected with N-CoR-flag and p65{Delta}TA in the absence (upper panel) or presence (lower panel) of LMB (10 ng/ml). (B) Immunofluorescence staining of 293T cells transfected with N-CoR-flag and p65{Delta}NES. N-CoR was detected with an {alpha}-flag antibody and a FITC-conjugated secondary antibody (left panels), {alpha}-p65 staining is represented in the middle panels corresponding to Cy3 signal, and nuclei were visualized by DAPI staining (right panels). (C) Hes1-luc transcriptional activity of NIH-3T3 cells cotransfected with N1-IC, N-CoR or/and p65{Delta}TA plasmids as indicated. Luciferase activity is represented as the fold induction relative to the basal level measured in cells transfected with empty vector. The average and standard deviation of duplicates from a representative experiment are presented. Lower panels show p65 protein levels. Protein loading for each lane was normalized according to ß-galactosidase expression.

 


View larger version (13K):

[in a new window]
  		Fig. 6. p65{Delta}TA increases the activity of promoters repressed by N-CoR. NIH-3T3 cells were contransfected with the above indicated luciferase reporters (A). N1-IC (0.5 µg) and N-CoR (1 µg) were contransfected in the presence or absence of p65{Delta}TA (0.5 µg). Fold activation was calculated relative to cells transfected with N1-IC alone. (B) Cotransfection of p65{Delta}TA (0.5 µg) with or without N-CoR (1 µg) expression vector. Cells were incubated for 12 hours in the presence of 20% FBS, and fold activation was calculated relative to cells transfected with the reporter alone. (C) Cells were transfected with a c-fos expression vector (0.5 µg), p65{Delta}TA (0.5 µg), and/or N-CoR (1 µg). Fold activation was calculated relative to cells transfected with c-fos alone.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2002