Fig. 1: PRMT5 interacts with and methylates G3BP2.
From: USP7- and PRMT5-dependent G3BP2 stabilization drives de novo lipogenesis and tumorigenesis of HNSC
A PRMT5 plasmid was transfected into HEK293 and Tu686 cells. After incubated for 48 h, cell lysates were measured by Immunoprecipitation (IP) assays using anti-PRMT5 beads, and the silver staining showed the ___location of PRMT5 and its associated protein G3BP2. Lane 1,3 for HEK293 cell. Lane 2,4 for Tu686 cell. B, C Co-immunoprecipitation (Co-IP) of PRMT5 and G3BP2 was performed in HEK293 cells. IgG was used as a negative control. D Association of PRMT5 with G3BP2 were verified by Co-IP assay with anti-Flag in Tu212 cells transfected with Flag-PRMT5 or Flag-G3BP2, respectively. E Schematic diagram showed the structure of PRMT5 (left) and the deletion constructs were co-transfected with Flag-G3BP2 into HEK293 cells. Cell lysates were precipitated with GST beads. G3BP2 was blotted with an anti-Flag antibody. Immunoblotting and Coomassie Brilliant blue staining were shown. F The methylation of G3BP2 was detected by western blot analysis using a custom-made methy-G3BP2 antibody in HEK293 and Tu212 cells transfected with or without PRMT5 plasmid. G Methylation site of G3BP2 was examined by liquid chromatography- mass spectrometry (LC-MS). R represents potential methylation site. H Sequences of the evolutionarily conserved residue R468 (red) in G3BP2. I Validation of R468 as the PRMT5-catalyzed G3BP2 methylation site in Tu212 and Tu686 cells. J In vitro methylation of G3BP2 in the presence of 3H-SAM. Recombinant GST-G3BP2-WT and G3BP2-R468K proteins were purified from bacteria and Flag-PRMT5 proteins were immunopurified from HEK293 cells. K Tu686 cells were treated with the indicated amounts of GSK3326595 for 24 h, protein levels of G3BP2 and methy-G3BP2 were assessed by western blot.
