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First published online 13 June 2006
doi: 10.1242/jcs.03001

Journal of Cell Science 119, 2797-2806 (2006)
Published by The Company of Biologists 2006
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Immunodetection of human telomerase reverse-transcriptase (hTERT) re-appraised: nucleolin and telomerase cross paths

Ying-Li Wu1,2, Charles Dudognon1, Eric Nguyen1, Josette Hillion1, Frédéric Pendino1, Ilona Tarkanyi3, Janos Aradi3, Michel Lanotte1, Jian-Hua Tong4, Guo-Qiang Chen2 and Evelyne Ségal-Bendirdjian1,*

1 INSERM U685, Hôpital Saint-Louis, Institut d'Hématologie, 1 avenue Claude Vellefaux, 75010 Paris, France
2 Department of Pathophysiology Key Laboratory of Cell Differentiation and Apoptosis of Ministry of Education, Shanghai Jiao-Tong University School of Medicine, Shanghai 200025, P. R. China
3 Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Nagyerdei krt. 98, 4012 Debrecen, Hungary
4 Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiao-Tong University School of Medicine, Shanghai 200025, P. R. China


Figure 1
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  		Fig. 1. hTERT is downregulated during NB4-LR1 differentiation. Differentiation (+) of NB4-LR1 cells was obtained by a 72-hour treatment with the combination of ATRA (1 µM) and 8-CPT-cAMP (200 µM). RNA and protein extracts were prepared as described in the Materials and Methods. hTERT mRNA expression was quantified by fluorescence real-time RT-PCR using the LightCycler® technology and the LightCycler TeloTAGGG hTERT Kit from Roche Diagnostics (Meylan, France). The hTERT level was normalized to the expression of the housekeeping gene porphobilinogen deaminase (PBGD) and expressed as a percentage of that detected in the untreated cells. Western blot analyses were performed using RCK-hTERT and NCL-hTERT antibodies. Positions of the molecular weight markers are indicated on the left. Note that the low molecular weight band on NCL-hTERT blot was not reproducibly detected.

 

Figure 2
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  		Fig. 2. Comparative study for the detection of hTERT protein in HeLa cells by immunoblotting and immunoprecipitation using commercially available antibodies. (A) Western blot analysis of total cell lysates from HeLa cells transfected with either HA-tagged hTERT (pB-hTERT-HA) or p-Babe empty vector alone (pB) using an anti-HA antibody and various anti-hTERT antibodies. (B) Immunoprecipitation (IP) of lysates from HeLa cells transfected with HA-tagged hTERT (pB-hTERT-HA), the non-tagged hTERT protein (pB-hTERT), or p-Babe empty vector alone (pB) with anti-HA antibody and immunoblotting with either anti-HA antibody or various anti-hTERT antibodies as indicated. Positions of the molecular weight markers are indicated on the left. Positions of the proteins of interest are indicated by arrows. Position of the heavy chain (Ig) of the anti-HA antibody is shown.

 

Figure 3
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  		Fig. 3. Detection of hTERT protein in NB4-LR1/hTERT (A) and GM847 cell extracts (B) using NCL-hTERT or RCK-hTERT antibodies. Western blot analyses of total cell lysates from NB4-LR1/hTERT (A) and from GM847 (B) cells transfected with either hTERT (GM-pB-hTERT) or pBabe empty vector alone (GM-pB). Positions of the molecular weight markers are indicated on the left. Positions of the proteins of interest are indicated by arrows.

 

Figure 4
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  		Fig. 4. Immunoprecipitations of lysates from NB4-LR1/hTERT cells with RCK-hTERT (A) or NCL-hTERT (B) antibodies and immunoblotting with both antibodies. Positions of the molecular weight markers are indicated on the left. Positions of the proteins of interest are indicated by arrows. Position of the heavy chain (Ig) of the NCL-hTERT antibody is shown. IgG, control for nonspecific binding by performing immunoprecipitation reaction using normal IgG. No immunoprecipitation was observed with control IgG.

 

Figure 5
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  		Fig. 5. Immunoprecipitations of lysates from NB4-LR1/hTERT cells with NCL-hTERT or nucleolin antibodies and immunoblotting with both antibodies. Positions of the molecular weight markers are indicated on the left. Positions of the proteins of interest are indicated by arrows. Position of the heavy chain (Ig) of antibodies is shown. IgG, control for nonspecific binding by performing immunoprecipitation reaction using normal mouse or rabbit IgG.

 

Figure 6
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  		Fig. 6. Identification of nucleolin and alpha actinin in NB4-LR1 cells by two dimensional electrophoresis and immunoblotting. NB4-LR1 proteins separated by isoelectric focusing in a pH 3-10 Bio-Rad IPG gel in the first dimension and by electrophoresis in an SDS 10% polyacrylamide gel in the second dimension. The proteins were blotted onto PVDF membranes and revealed using the indicated antibodies. Positions of the molecular weight marker are indicated on the left. IP, isoelectric point.

 

Figure 7
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  		Fig. 7. Immunofluorescence and confocal microscopy. (A) hTERT was detected in NB4-LR1 cells using RCK-hTERT (red; Alexa Fluor 594-conjugated anti-rabbit antibody). Nucleolin was detected either by MBL-nucleolin (green; Alexa Fluor 488-conjugated anti-mouse antibody) or by SC-nucleolin (red; Alexa Fluor 594-conjugated anti-rabbit antibody). NCL-hTERT antibody detected a protein (green; Alexa Fluor 488-conjugated anti-mouse antibody) that colocalized with nucleolin. Nuclei are stained in blue with DAPI. Panels f and l show the superimposition of RCK-hTERT, NCL-hTERT, DAPI signals and SC-nucleolin, NCL-hTERT and DAPI signals, respectively. (B) Higher magnification of two NB4-LR1 cells stained using either RCK-hTERT (b) or NCL-hTERT (d). Panels c, e and f are superimposed images of DAPI and RCK-hTERT or NCL-hTERT signals and RCK-hTERT and NCL-hTERT signals, respectively. (C) Staining of GM847 cells using either RCK-hTERT (red; Alexa Fluor 594-conjugated anti-rabbit antibody) or NCL-hTERT (green; Alexa Fluor 488-conjugated anti-mouse antibody) antibodies. Nuclei are stained blue with DAPI. Bars, 10 µm.

 

Figure 8
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  		Fig. 8. Nucleolin protein in NB4-LR1 cells after differentiation and apoptosis. (A) Differentiation (+) of NB4-LR1 cells was obtained by a 72-hour treatment with a combination of ATRA (1 µM) and 8-CPT-cAMP (200 µM). (B) Apoptosis was induced either by a 24-hour treatment with oridonin (4 µg/ml) or a 24-hour treatment with As2O3 (5 µM). Proteolytic fragments are indicated.

 

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© The Company of Biologists Ltd 2006