Figure 4

CARM1 and methylated HuR reduce replicative senescence. (A) Western blot analysis of CARM1, HuR, and GAPDH protein levels in early-passage (Young, ~27 pdl, Y) and late-passage (Senescent, ~60 pdl, S) 2BS cells. (B) HuR was immunoprecipitated from the whole cell lysates (100 μg) described in Figure 3A, whereupon the total or methylated HuR was assessed by Western blot analysis using HuR or M/DMA antibodies, respectively. Western blotting signals were quantified by densitometry. Data are representative from 3 independent experiments. (C) Cytoplasmic extracts prepared from cells described in Figure 4A 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 HuR by Western blotting. A 10-μg aliquot of whole-cell lysates (Input) and binding of GAPDH to the cyclin A, cyclin B1, c-fos, SIRT1, and p16 3′UTR were also tested. (D) Cytoplasmic extracts described in Figure 4C were subjected to RNP IP assays using anti-flag antibody, the presence of cyclin A, cyclin B1, c-fos, SIRT1, and p16 mRNAs in the IP materials were assessed by real-time qPCR.