TTP functions in the nucleus.

<p>(A) Immunofluorescence staining. Plasmids encoding the Flag-TTP constructs listed in the figure were transfected into HeLa cells, and the protein products were immunostained with anti-Flag and anti-PABPN1. Flag-TTP constructs are stained green and endogenous PABPN1 is stained red. The blue signals are those of DAPI, which locate nuclei. (B) Co-immunoprecipitation of Flag-TTP constructs and PABPN1. HEK293T cells were transfected with plasmids encoding the indicated Flag-TTP constructs. After expression, the Flag constructs were immunoprecipitated from cell lysates with anti-Flag M2 agarose. Co-immunoprecipitated PABPN1 was detected by western blotting with anti-PABPN1. (C) Reporter assay using TNFα-ARE-containing luciferase plasmid. HEK293T cells were co-transfected with 0.5 µg of TNFα-ARE-containing luciferase plasmid or the control luciferase reporter plasmid and 0.1 µg of the WT Flag-TTP, 0.15 µg of Flag-TTP@15-319 or Flag-TTP@15-306 or Flag-TTP@15-186, 0.18 µg of Flag-TTP@1-306 and 0.22 µg of Flag-TTP@1-186 deletion mutant expression plasmid and 0.5 µg of the pRenilla-luciferase control plasmid. The protein expression levels were detected by western blotting with anti-Flag and the loading control anti-Tubulin in the lower panel. Duplicate reactions were each run three times. For each sample, its luciferase activity was first divided by its renilla luciferase activity and then normalized to the control firefly/renilla luciferase value. *, p<0.05; ****, p<0.001.