Figure 4.

CARM1 can be co-immunoprecipitated with UPF1 and the interaction between UPF1 and the β-Globin T39 mutant (MT) decreases with CARM1 knockdown. (A) Total cell lysates were prepared from MN-1 cells and subjected to immunoprecipitation with an IgG CTRL or UPF1 antibodies. Immunoprecipitated proteins were then analysed by western blot using antibodies against UPF1 and CARM1. (B) UPF1 immunoprecipitation experiments were performed with or without (w/o) pretreatment of the cell lysate with RNase A (1 μg/ml) for 30 min at 37°C. (C) Then, the CARM1/UPF1 ratios in response to the RNase A treatments were assessed. Values shown in the bar graph are means +/− SEM (n = 3). (D) The MT reporter was transiently transfected either into the MN-1 pGIPZ CTRL or the MN-1 shCARM1 cell line. RT-PCR analysis was performed using primers specific for the MT mRNA or pre-mRNA, on total RNA extracted from the CTRL (left panel) or shCARM1 (right panel). (E) β-Globin mRNA levels, shown here as percent bound to UPF1, were normalized to overall immunoprecipated UPF1 levels and Gapdh mRNA was used as a loading control. Data are means +/− SEM (n = 3).