(A) WB for RBM15 in 293T cells overexpressing PRMT1 V1 and V2. PRMT1 V2 was detected by anti-V2 specific antibody (PRMT1 V2). PRMT1 V1 and V2 were detected by an antibody for all isoforms (labeled as PRMT1). (B) The level of the RBM15 protein as detected by WB in MEG-01 cells treated with methyltransferase inhibitors (Adox and MTA mix) or DB75. (C) RBM15 protein level was measured by WB in MEG-01 cells with two doxycycline-inducible shRNA against PRMT1 (on the left). In the middle and right sides are real-time PCR results to show the mRNA levels of total amount of PRMT1, PRMT1 V2, and RBM15 in shPRMT1#1 stable MEG-01 cell line (middle) and in shPRMT1#2 stable MEG-01 cells (right). All data are presented as mean ± standard deviation from three independent experiments. (D) RBM15 protein level was measured by WB in MEG-01 cells induced by Dox to express PRMT1 V2 isoform. On the right are the real-time PCR charts for PRMT1 V2 and RBM15 mRNA levels. Data are presented as mean ± standard deviation from three independent experiments. (E) RBM15 protein level was accessed by WB in a MEG-01 stable cell line expressing shRNA against V2. The names of antibodies are listed on right. The pRS vector retrovirus infected MEG-01 cells were used as control. (F) WB with anti-Flag antibody to detect the protein levels of RBM15 wild type and R578K mutant proteins in 293T cells overexpressing PRMT1 V2 and RBM15 proteins. (G) The half-life of the RBM15 proteins in MEG-01 cells, and stable cell lines overexpressing Flag-tagged RBM15 and RBM15 R578K were assessed by WB. Cyclohemixide were added to stop protein synthesis 30 min before harvesting cells as the 0 time point. The half-life curves were plotted by GraphPad Prism 6. Adox, adenosine dialdehyde; DMSO, dimethyl sulfoxide; Dox, doxycycline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; MTA, methylthioadenosine; PCR; polymerase chain reaction; PRMTs, protein arginine methyltransferases; shRNA; short hairpin RNA; WB, western blot.