Figure 3

Influences of CARM1-mediated methylation on the subcellular distribution and RNA-binding affinity of HuR. (A and B) Western blot analysis was performed to assess the presence of endogenous HuR (A) as well as flag-tagged HuR (flag-HuR and flag-HuRΔ) (B) in whole-cell (Total, 10 μg), cytoplasmic (Cyto., 40 μg), and nuclear fractions (Nuc., 5 μg) prepared from cells described in Figure 1A and 1D. Assessment of the levels of cytoplasmic-specific tubulin and nuclear-specific HDAC1 served to verify the quality and equal loading of the cytoplasmic and nuclear preparations, respectively. (C) Cytoplasmic extracts (100 μg) described in Figure 3A were subjected to RNA pull-down assays using biotinylated 3′UTR fragments of cyclin A, cyclin B1, c-fos, SIRT1, and p16 to detect bound endogenous HuR by Western blotting. A 10-μg aliquot of whole-cell lysates (Lys.), binding of HuR and GAPDH to the beads (Neg.), and binding of GAPDH to the cyclin A, cyclin B1, c-fos, SIRT1, and p16 3′UTR were also tested. (D) Cytoplasmic extracts (100 μg) described in Figure 3A were subjected to RNP IP assays using anti- HuR antibody. The presence of cyclin A, cyclin B1, c-fos, SIRT1, and p16 mRNAs in the IP materials were assessed by real-time qPCR. (E, F) Cytoplasmic extracts (100 μg) described in Figure 3B were either subjected to RNA pull-down assays (E) or RNP IP assays (F) to assess the association of flag-HuR and flag-HuRΔ with the mRNAs of cyclin A, cyclin B1, c-fos, SIRT1, and p16, as described in Figure 3C and 3D. (G, H) Cytoplasmic extracts described in Figure 3A and 3B were either used for RNA pull-down assays (G) or used for RNP IP assays (H) to assess the association of AUF1 with p16 mRNA, as described in Figure 3C and 3D.