Fig. 7. MCPIP3 directly degrades IL-6 and Regnase-1 mRNAs.
a–c 293T cells were co-transfected with Regnase-1 (a) or MCPIP3 (b, c), and IL-6 CDS with 3′ UTR (a, b) or without 3′ UTR (c). After 48 h, IL-6 in supernatants were measured by ELISA (n = 4 replicates for IL-6 CDS only; n = 3 replicates for all other groups). d Degradation of Il6 mRNA by MCPIP3 was measured by a luciferase reporter system. 3′ UTR of Il6 were inserted downstream of the luciferase gene. These reporter plasmids were then transfected into 293T cells, along with empty, MCPIP3 or mutant MCPIP3 overexpression plasmids (n = 4 replicates). e 3′ UTR of negative control genes (β-actin, Hprt, Ccr7) were inserted downstream of the luciferase gene. f 293T cells were co-transfected with MCPIP3 and IL-6 CDS with 3′ UTR. After 48 h, Il6 mRNA in transfected cells were measured by qPCR (n = 3 replicate wells). g Zc3h12a (the gene encoding Regnase-1) expression in sorted Zc3h12c+/+/Zc3h12c−/− bone marrow-derived macrophages (BMDMs) was measured by qPCR (n = 3 mice for Zc3h12c+/+; n = 4 mice for Zc3h12c−/−). h Zc3h12a expression in purified Zc3h12cfl/fl/Zc3h12cMΦΔ peritoneal macrophages (Per. MΦ) was measured by qPCR (n = 3 mice for Zc3h12c+/+; n = 4 mice for Zc3h12c−/−). i Zc3h12a expression in sorted Zc3h12cfl/fl/Zc3h12cDCΔ bone marrow (BM) pDCs was measured by qPCR (n = 4 mice per group). j Degradation of Zc3h12a mRNA by MCPIP3 was measured by a luciferase reporter system (n = 3 replicates). k 293T cells were transfected with FLAG-tagged MCPIP3 together with MYC-tagged Regnase-1. After 48 h, whole cell lysates were immunoprecipitated with anti-MYC or FLAG antibodies. Gray area quantification of the Regnase-1-MYC was calculated by ImageJ, after normalizing with β-actin bands (n = 3 replicate bands). The samples were derived from the same experiment and that gels/blots were processed in parallel. Data are representative of at least three independent experiments (mean ± S.D.; two-tailed Student’s t test). Source data are provided as a Source Data file.