Fig. 4: Interaction between HuD and 3′UTR of Ccl2 mRNA. | Cell Death & Disease

Fig. 4: Interaction between HuD and 3′UTR of Ccl2 mRNA.

From: Loss of RNA binding protein HuD facilitates the production of the senescence-associated secretory phenotype

Fig. 4

A The interaction between HuD and Ccl2 mRNA was confirmed by RNA IP followed by RT-qPCR using anti-HuD and control IgG antibodies. Gapdh mRNA was used for normalization. B Top: A schematic of mouse Ccl2 mRNA (NM_011333.3). The UTRs of Ccl2 mRNA (5U and 3U) were transcribed in vitro using T7 RNA polymerase and biotin-labeled nucleotides. Bottom: The biotinylated transcripts (Ccl2 5U, Ccl2 3U, and GAPDH 3U) were incubated with the lysate from N2a cells. The proteins binding to the transcripts were pulled-down using streptavidin magnetic beads and analyzed via western blotting using HuD antibody. Biotinylated GAPDH 3U was used as a negative control. C Schematic diagram of EGFP reporters. The reporter plasmid (pEGFP-Ccl2 3U) was constructed by inserting the 3′UTR of Ccl2 mRNA (533–806 nt) into the pEGFP-C1. (▼: stop codon). D After sequential transfection with siRNAs and EGFP reporter plasmids, relative fluorescence of EGFP from each sample was measured by fluorescence microscopy and the levels of HuD and β-ACTIN were assessed by western blotting analysis. Images are representative, and data indicate the mean ± SEM from three independent experiments. The statistical significance of the data was analyzed via Student’s t-test; *p < 0.05; **p < 0.01.

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