A computer-assisted two-dimensional gel electrophoresis approach for studying the variations in protein expression related to an induced functional repression of NFkappaB in lymphoblastoid cell lines.
Strategies are needed for conclusive interpretation of two-dimensional gel electrophoresis (2-D PAGE) maps in order to identify pertinent differences in protein expression during regulation of the transcription of discrete sets of genes. The model used in this study was a human lymphoblastoid cell line in which a functional repression of the transcription factors NFkappaB was obtained by induction of overexpression of IkappaBalpha, a physiological inhibitor of NFkappaB. The analytical methodology used relies on the comparison of two sets of 2-D PAGE maps for detecting differences in protein expression between samples overexpressing or not overexpressing IkappaBalpha. The analysis was based on a combination of an automatic computerized analysis, constituting an actual aid for deciding, and of an interactive visual validation, corresponding to the interpretation of computer propositions. This strategy is proposed as a rapid way to detect potential variations in protein expression applicable to any biological model. In this study, correspondence analysis data made it possible to discrimate between the samples overexpressing or not overexpressing IkappaBalpha, and pointed out some of the potential meaningful spots characterizing the samples in which NFkappaB was active. Then, after visual validation of the computer data, 53 polypeptides were considered to be different in the two classes of gels. Five polypeptides were specifically found in both samples overexpressing IkappaBalpha. The overexpression of IkappaB also induced a lower expression of 11 polypeptides. Finally, 15 polypeptides were only expressed in samples in which IkappaBalpha was not overexpressed and, consequently, in which NFkappaB factors were active. Thus, these polypeptides are candidates for further analysis as putative target gene products of NFkappaB.
Joubert-Caron
R
R
Biochimie Cellulaire des Hémopathies Lymphoïdes et des Vascularites, UFR Léonard de Vinci, Bobigny, France. caron@smbh.univ-paris13.fr
Feuillard
J
J
Kohanna
S
S
Poirier
F
F
Le Caër
J P
JP
Schuhmacher
M
M
Bornkamm
G W
GW
Polack
A
A
Caron
M
M
Bladier
D
D
Raphaël
M
M
D000180:Acrylic Resins; D001331:Automation; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D014644:Genetic Variation; D006801:Humans; D020672:I-kappa B Proteins; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured
D000180:Acrylic Resins; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C016680:polyacrylamide gels; D000072000:NF-KappaB Inhibitor alpha