Figure 6: Zinc ions affect the nucleotide-binding ability of PCBP1 and sortilin mRNA stability. | Scientific Reports

Figure 6: Zinc ions affect the nucleotide-binding ability of PCBP1 and sortilin mRNA stability.

From: TLR signals posttranscriptionally regulate the cytokine trafficking mediator sortilin

Figure 6

(a) EMSA analysis of PCBP1. 32P-labeled poly-C oligonucleotide probe (100 nM) was mixed with buffer alone (P) or yeast lysates expressing FLAG-PCBP1. Samples were separated by PAGE. A 10-fold molar excess of unlabeled poly-C oligonucleotide and mutated oligonucleotide was added as specific competitor (S) and nonspecific competitor (N), respectively. Zinc sulfate and ferrous ammonium sulfate (both 100 μM) with or without 10 mM EDTA were incubated with samples prior to incubation with the probes. (b) Quantification of sortilin mRNA after supplementation with zinc sulfate and the ionophore pyrithione. Macrophages were treated with zinc sulfate and pyrithione (Zn/P) for 6 h and then harvested for isolation of total RNA. Total RNAs were subjected to qRT-PCR to quantify sortilin mRNA. Data are mean ± SD (n = 3). *p < 0.01 versus control. (c) Zinc chelation interferes with destabilization of sortilin mRNA by zinc. Macrophages were treated with 2 μM Zn/P with or without various concentrations of TPEN for 6 h. Total RNAs were subjected to qRT-PCR to quantify sortilin mRNA. Data are mean ± SD (n = 3). *p < 0.01. (d) Zinc chelation does not prevent CpG-B-dependent Sortilin mRNA degradation. Sortilin mRNA was quantified after 1 μM CpG-B stimulation for 6 h with or without 2 μM TPEN. Data are mean ± SD (n = 3).

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