Mapping and identification of interferon gamma-regulated HeLa cell proteins separated by immobilized pH gradient two-dimensional gel electrophoresis.
Interferon gamma (IFN-gamma) is a potent immunomodulatory lymphokine, secreted by activated T-lymphocytes and NK-cells during the cellular immune response. Actions of IFN-gamma are mediated through binding to the IFN-gamma-receptor, present on most cells, and the subsequent activation of a great magnitude of IFN-gamma responsive genes has been reported previously. Our goal is to identify and map IFN-gamma-regulated HeLa cell proteins to the two-dimensional polyacrylamide gel electrophoresis with the immobilized pH gradient (IPG) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) system. A semiconfluent layer of HeLa cells was grown on tissue culture plates, and changes in protein expression due to 100 U/mL IFN-gamma were investigated at different periods after treatment, using pulse labeling with [35S]methionine/cysteine in combination with 2-D PAGE (IPG). The identity of eight protein spots was elucidated by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), and several variants of the IFN-gamma-inducible tryptophanyl-tRNA synthetase (hWRS) were detected by immunoblotting.
Shaw
A C
AC
Department of Medical Microbiology and Immunology, University of Aarhus, Denmark. shaw@medmicro.aau.dk
Røssel Larsen
M
M
Roepstorff
P
P
Justesen
J
J
Christiansen
G
G
Birkelund
S
S
D000595:Amino Acid Sequence; D017305:Annexin A1 / Q000032:analysis; D003545:Cysteine; D003546:Cysteine Endopeptidases; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007371:Interferon-gamma / Q000276:immunology* / Q000494:pharmacology; D007633:Keratins / Q000032:analysis; D008715:Methionine; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013462:Sulfur Radioisotopes; D014369:Tryptophan-tRNA Ligase / Q000032:analysis; D015854:Up-Regulation
D017305:Annexin A1; D011506:Proteins; D013462:Sulfur Radioisotopes; C071313:LMP-2 protein; D007633:Keratins; D007371:Interferon-gamma; D008715:Methionine; D003546:Cysteine Endopeptidases; D014369:Tryptophan-tRNA Ligase; D003545:Cysteine