A Northwestern blotting approach for studying iron regulatory element-binding proteins

At least two proteins binding to iron regulatory elements (IRE) in mRNA are known, designated as iron regulatory proteins (IRP) 1 and 2. Their binding activity is widely studied by electrophoretic mobility shift assays (EMSA), which resolves one or two bands depending on the species. We used Northwestern blotting to resolve this EMSA complex into four components, and identified two other IRE-binding peptides present in HepG2 cell extracts. We designate these six peptide bands A to F on Northwestern blots, ranging in apparent molecular weight from 111 to 37 kDa. Band C is lost when cells are preloaded with iron or when leupeptin (but not several other protease inhibitors) is included in the extraction buffer. Band E is also lost with leupeptin but increases with iron loading. Binding of all bands is sensitive to iron in vitro. Two-dimensional electrophoresis reveals additional processing, especially indicating charge variants of band C. Northwestern bands A and B both react with an antibody to IRP-1 on parallel Western blots. We conclude that cellular processing can produce multiple IRE-binding species that may be involved in a more complex regulation of iron metabolism than generally appreciated. The Northwestern approach should facilitate studies of processing and binding requirements of proteins and peptides that recognize the IRE sequence.