FIGURE 1:

Identification of PRMT5 as an ASK1-binding protein. (A) EAhy926 cells were transfected with pFLAG-CMV-ASK1 and then treated with 100 ng/ml VEGF for 30 min. FLAG-tagged ASK1 protein was purified by anti-FLAG agarose. Purified proteins were electrophoresed and stained with Coomassie brilliant blue. PRMT5 (72 kDa) is indicated in the gel. (B) The 72-kDa band was cut and in-gel digested with trypsin. Peptides were subject to LC-MS/MS assay. Identified peptides that matched with the sequence of PRMT5 are shown in yellow. MS/MS spectrum of the tryptic peptide YSQYQQAIYK. Fragment ions are indicated as b and y ions. (C) Cell lysates from A were immunoprecipitated with anti-FLAG antibody or control immunoglobulin G (IgG) and then separated by 8% SDS–PAGE. Transferred membrane was immunoblotted with either anti-FLAG or PRMT5 antibody.