Fig. 2

Identification of GAPDH as a novel substrate of PRMT3. a Coomassie blue of GFP-purified protein complexes from GFP- and GFP-PRMT3-ovexpressing PANC-1 cells. PRMT3-interacting proteins were pulled down and analyzed by mass spectrometric analysis. b The PRMT3-interacting metabolic enzymes identified in a previous study [19], and this study is shown and three common proteins are labeled by red color. c Cell lysates were collected from GFP- and GFP-PRMT3-overexpressing PANC-1 cells and subjected to immunoprecipitation using the GFP and GAPDH antibodies followed by western blotting to detect GAPDH and GFP. d Left upper panel, GAPDH proteins were immunoprecipitated from GFP- and GFP-PRMT3-overexpressing PANC-1 cells and subjected to western blotting to detect the level of asymmetric dimethylated arginine (ADMA). Right upper panel, L3.6pl cells were treated with SGC707 (100 μM) for 48 h. GAPDH proteins were immunoprecipitated, followed by western blotting to detect the level of ADMA. Bottom lower panel, L3.6pl cells were transfected with PRMT3-targeting shRNAs for 48 h. GAPDH proteins were immunoprecipitated and subjected to western blotting to detect the level of ADMA. e GAPDH proteins were purified from GFP-PRMT3-overexpressing PANC-1 cells using GAPDH antibody, and the immunoprecipitated complexes were separated by SDS-PAGE. The protein bands corresponding to GAPDH were excised and subjected to mass spectrometric analysis. f Alignment of the amino acid sequences around R248 of GAPDH protein in different species