Fig. 3

PRMT3-mediated R248 methylation increases the catalytic activity of GAPDH. a Left panel, the GAPDH activity of GFP-PRMT3-overexpressng PANC-1 cells treated with or without specific PRMT3 inhibitor SGC707 (100 μM) was determined by GAPDH activity kit. Right panel, the GAPDH activity of Miapaca-2 cells with or without PRMT3-targeting shRNA was determined by GAPDH activity kit. Error bars, SEM. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. b Flag-tagged wild-type GAPDH (WT) and R248K mutant expression vectors were transfected into HEK293T cells (left) and L3.6pl cells (right), respectively. After 48 h, the GAPDH activity was measured by ELISA assays and the expression levels of GAPDH were detected by western blotting. Error bars, SEM. n = 3. *p < 0.05, **p < 0.01. c HEK293T cells were co-transfected with pcDNA3, Flag-PRMT3, Flag-tagged GAPDH-WT, or R248K mutant expression vectors. After 48 h, the GAPDH activity was measured by ELISA assays and the levels of expressed proteins were detected by western blotting. Error bars, SEM. n = 3. **p < 0.01, ***p < 0.001. d Flag-tagged wild-type GAPDH (WT) and R248K mutant expression vectors were transfected into HEK293T cells. After 48 h, the cells were treated with cycloheximide (10 μg/ml) and cellular proteins were harvested at the indicated time points. The levels of Flag-tagged GAPDH were investigated by western blotting, and actin was used as an internal control. e Cell lysates were extracted from the indicated stable cell lines and subjected to 10% native gel electrophoresis (left) and SDS-PAGE (right), respectively. Overexpression of PRMT3 significantly enhanced the tetramer formation of wild-type GAPDH proteins but not R248-mutant GAPDH proteins