Figure 3.

TTP (encoded by ZFP36), represses NLRP3 3′-UTR luciferase reporters and binds to the long NLRP3 3′-UTR isoform. a, HEK293T cells were transfected with an empty vector or full-length NLRP3 3′-UTR firefly luciferase reporter constructs, an internal TK-Renilla control, and ZFP expression vectors. Firefly luciferase readings were normalized to Renilla activity and the ratio between the expression of the NLRP3 3′-UTR and empty vector in two independent experiments was plotted against each other. Because most data points scatter around 1, outliers represent potential regulators of NLRP3 3′-UTR expression. Zfp36 is highlighted. b, NLRP3 3′-UTR vectors described in the legend to Fig. 1c were co-expressed with an empty vector (pcDNA3.1), wild-type (wt), or zinc finger mutant TTP (mut) in HEK293T cells. Renilla luciferase expression 24 h after transfection was normalized to Firefly luciferase activity. Relative expression normalized to psiCHK-2 for each expression vector and pcDNA ± S.E. of 6 independent experiments is shown. c, TTP RNA-IP from lysate of THP-1 cells stimulated with PMA for 4 h. RNA was extracted from the pulldown and GAPDH, NLRP3, and NLRP3′long (long 3′-UTR isoform) and IL1B were quantified by qPCR using SYBR primers. Enrichment in TTP over IgG control pulldown ± S.E. of three independent experiments is shown. d, RNA-IP samples were analyzed by 3′-RACE for NLRP3 and ACTB. PCR products were visualized by agarose gel electrophoresis and band intensities were quantified. Relative intensity of each isoform relative to IgG is shown. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.