A new enzyme-linked immunosorbent assay system against the <i>N</i>-terminal propiece of interleukin-1α

Abstract

is produced inside cells in its precursor form . Enzymatic cleavage yields mature (mIL-1α) and the propiece of , which are thought to be localized in the nucleus, because of the presence of nuclear localizing signals. Studies of ppIL-1α function have been hampered by the lack of a ppIL-1α-specific antibody (Ab). In the present study, the authors generated anti-ppIL-1α Ab by using recombinant histidine-tagged ppIL-1α (His-ppIL-1α) as an immunogen. Rabbits were immunized with His-ppIL-1α, and affinity-purified Ab was obtained. Ab reactivity and specificity were examined by Western blotting. The antibody successfully recognized transfectant-derived green fluorescence protein (GFP)-tagged ppIL-1α but not GFP. A sandwich enzyme-linked immunosorbent assay (ELISA) system established by biotinylating the anti-ppIL-1α Ab successfully detected GFP-ppIL-1α. The Ab and ELISA system allows functional analysis of ppIL-1α and improves understanding of ppIL-1α.

Analysis of the effects of importin α1 on the nuclear translocation of IL-1α in HeLa cells

“…Using the Quick-Change site-directed mutagenesis kit (Agilent, Böblingen, Germany), expression vector (HiBiT-IL-1α-His) containing the N-terminal HiBiT-tag (11 amino acids [VSGWRLFKKIS]) and the C-terminal His-tag was constructed, and pcDNA-IL-1α vector was used as a template 64 . The cNLS-deletion mutant of IL-1α (∆NLS) was constructed using the HiBiT-IL-1α-His vector as a template and the above-mentioned kit.…”