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Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization
Published on: October 6, 2019
Identification of proteins immunologically related to interferon regulatory factor-1 that bind with interferon
Sara Contente1, Frank A Attard, Robert M Friedman
1Department of Pathology and United States Military Cancer Institute, Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA. scontente@usuhs.mil
Abstract:
Interferon regulatory factor (IRF)-1 expression was surveyed in nontransformed and oncogene-transformed mouse fibroblasts, using Western immunoblot with an IRF-1-specific antiserum, to examine possible differences resulting from cellular transformation. Ten additional proteins that reacted with the IRF-1 antibody and that underwent specific competition by peptide antigen were observed in extracts of both nontransformed and oncogene-transformed cell lines. Cross-reacting proteins were also observed in mouse macrophage extracts. Protein was captured from fibroblast nuclear extracts, using oligonucleotides representing IRF-binding sequences linked to magnetic beads. Captured proteins were eluted and analyzed by immunoblot with anti-IRF-1. Along with 43-kDa IRF-1, 4 of the 7 nuclearly located cross-reacting proteins (97, 90, 66, and 33 kDa) were found to complex with the IRF binding element. These proteins, with an epitope in common with the IRF-1 C-terminal region and IRF element DNA sequence-binding capability, may represent new members of the IRF family.
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