Figure 7.
Binding of cytoplasmic TIAR, AUF1, and HuR to ZO-1 transcripts in cells described in Figure 6. (A) Schematic representation of ZO-1 mRNA and the AU-rich sequences in its 3′-UTR. (B) Representative immunoblots of TIAR, AUF1, and HuR using the pull-down materials from the 3′-UTR (a) or CR (b). Cytoplasmic lysates (120 μg each) prepared from either controls and JunD-transfected cells were incubated with 6 μg of biotinylated ZO-1 3′-UTR or CR for 30 min at 25°C, and the resulting RNP complexes were pulled down by using streptavidin-coated beads. Levels of TIAR, AUF1, and HuR proteins in the pull-down materials were measured by Western blot analysis. Three experiments were performed that showed similar results. (C) Association of endogenous TIAR with endogenous ZO-1 mRNA after ectopic expression of the junD gene. a, lysates from controls and JunD-transfected cells were used for IP in the presence of anti-TIAR antibody or nonspecific IgG1. RNA in the IP material was used in RT-PCR reactions to detect the presence of ZO-1 mRNA and the resulting PCR products (∼249 base pairs) were visualized in agarose gels. b, quantification of ZO-1 mRNA levels in the IP materials as measured by real-time PCR analysis. Values are the means ± SE of data from three separate experiments. *p < 0.05 compared with controls.