Figure 4. Silencing of PRMT1 enhances SMN-FMRP interaction.
(A) Expression of arginine methyltransferases (PRMT1-8) was silenced using siRNA pools by reverse transfection in HEK293T cells, which were transfected with FLAG-FMRP (labelled F-FMRP) the next day. α-FLAG IPs were analyzed by immunoblotting using α-SMN. The red arrow (→) indicates the endogenous SMN. Input protein levels of SMN and FLAG-FMRP were assessed with α-SMN and α-FLAG, respectively. Silencing efficiency was assessed for PRMT1 and PRMT5 (bottom panels). (B) As in panel (A), but only PRMT1 and PRMT5 were silenced. (C) HEK293T cells were cultured on a larger scale, proteins were extracted, and co-immunoprecipitation assays were performed on endogenous proteins. (D) Polysome fractions were analyzed by sucrose gradient from mouse MN1 cholinergic motor neuron cell extracts. On the left, MgCl2-treated (control), and on the right, RNase A-treated (100 µg/ml) samples. Whole-cell extracts were loaded in the first lane.