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. 2016 Dec 26;8(32):52432–52444. doi: 10.18632/oncotarget.14194

Figure 5. Stabilization of IL-8 mRNA by CRABP-II/HuR complex.

Figure 5

A. Schematic diagram of human IL-8 mRNA sequence. 9 putative AU rich elements (ARE) are shown in the 3′UTR region. B. Interaction between CRABP-II and HuR in Panc-1 cells. The cell lysates were pre-treated with 100 μg/ml RNase for 30 min. Immunoprecipitation was performed by using anti-HuR mAb (3A2) and mouse IgG was used as negative control. Anti-CRABP-II antibody was used for immunoblot. C. RNA immunoprecipitation (RIP) showing the enrichment of CRABP-II on IL-8 mRNA. Protein-RNA complexes were precipitated by anti-flag beads. RNA was isolated from beads and the binding of IL-8 mRNA was assessed by qRT-PCR. Actin mRNA was used as control. RNA isolated from 10% of input whole cell lysis was used as input control. Data represent the means ± SD of three independent experiments. **p<0.01 by student t-test. D. Half-life of IL-8 mRNA in CRABP-II knockout and control cells. Cells were pre-cultured in charcoal stripped media for 48 hrs followed by treatment with actinomycin D. RNA was isolated at denoted time points and the mRNA of IL-8 was assessed by qRT-PCR. Data represent the means ± SD of triplicate wells from three independent experiments. **, p<0.01 by one-way ANOVA test.