High-performance affinity purification for identification of 15-deoxy-Δ12,14-PGJ2 interacting factors using magnetic nanobeads

Prostaglandin J(2) (PGJ(2)) family have been reported to show various kinds of biological activities. Considerable progress has been made toward understanding the mechanism of adipogenesis, however, the mechanisms of other actions of PGJ(2) family remain controversial. The 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) is one of the members of PGJ(2) family, and is known as a ligand for peroxisome proliferator-activated receptor g (PPAR gamma), which promotes the expression of the crucial genes for adipogenesis. In this study, we found that 15d-PGJ(2) did not stimulate PPAR gamma-mediated gene expression in HEK293 cells whereas 15d-PGJ(2) transactivated PPAR gamma-dependent transcription in other cell lines. Moreover, we confirmed that 15d-PGJ(2) suppressed the growth of HEK293 cells. These observations suggest that 15d-PGJ(2) shows another biological activity e. g. growth inhibition in HEK293 cells via unknown receptor for 15d-PGJ(2). The aim of this study is to develop and validate effective purification system for PGJ(2) interacting factors (PGJIFs). We have recently developed high performance magnetic nanobeads. In this study, we have newly developed 15d-PGJ(2)-immobilized beads by conjugating 15d-PGJ(2) to the surface of these nanobeads. Firstly, we showed that PPARg specifically bound to 15d-PGJ(2)-immobilized beads. Secondly, we newly identified voltage dependent anionic channel 1 (VDAC1) as new PGJIF from crude extracts of HEK293 cells using this affinity purification system. These data presented here demonstrate that 15d-PGJ(2)-immobilized beads are effective tool for purification of PGJIFs directly from crude cell extracts. Copyright (c) 2010 John Wiley & Sons, Ltd.