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Figure 2. IL-10 inhibits HuR binding to TNF-3'UTR ARE sequences in RNA EMSAs. A) Representative RNA-EMSA. Cytoplasmic extracts from variously treated U937 cells were made and allowed to react with a radio-labeled ([
-32P]-uridine triphosphate) single-stranded RNA fragment corresponding to the sense orientation of the 3'UTRARE of the human TNF mRNA prepared by in vitro transcription (IVT). For competition, indicated fold excess of unlabeled IVT probe were added 20 min prior to the addition of radioactive probe (Lanes: 1, Untreated; 2, LPS; 3, interleukin-10; 4, LPS+interleukin-10; 5, LPS+25X, 3'UTRARE cold probe; 6, LPS+50x3'UTRARE cold probe; 7, LPS+25x3'UTRAREmut cold probe; and 8, LPS+50x3'UTRAREmut cold probe). B) For super shift of observed complexes, anti-HuR, anti-TTP antibodies, and control IgG antibodies were mixed in the EMSA reaction before the addition of radiolabeled probe. Anti-HuR supershifted IL-10-sensitive complex II, whereas anti TTP diminished IL-10 insensitive complex III. C) Total protein extracts from cells treated as indicated were subjected to Western blot analysis for known ARE binding proteins, HuR, and TTP. IL-10 suppressed LPS-induced expression of HuR.
