Fig. 1: GLUD1 is methylated at arginine 76.
From: AKT1 phosphorylates PRMT7 to promote GLUD1 methylation and gastric cancer progression
A HEK 293T cells expressing Flag-tagged GLUD1 were treated with 20 μM or 50 μM AdOx for 24 h. Western blot analysis of GLUD1 methylation using a mono-methylarginine (Rme1) antibody, following immunoprecipitation with Flag beads. B HEK 293T cells were transfected with wild-type GLUD1 or methylation-deficient mutants (R76K, R496K) GLUD1. Western blot analysis of GLUD1 methylation after immunoprecipitation with Flag beads. C GLUD1 R76 is evolutionarily conserved. Amino acid sequences alignment of R76 across six mammalian species. D HEK 293T cells expressing either GLUD1 WT or R76K mutant were treated with or without 20 μM AdOx for 24 h. Western blot analysis of GLUD1 methylation following immunoprecipitation with Flag beads. E A site-specific methylation antibody (meGLUD1[R76]) specifically recognized the R76 mono-methylated (R76me1) peptide but not the unmodified peptide with dot blot assays. F The R76me1 peptide blocks the meGLUD1(R76) antibody but not the unmodified peptide. After incubation with unmodified peptide or R76 mono-methylated peptide (R76me1), meGLUD1(R76) antibody was used to detect immunopurified GLUD1. G AGS and MFC cells were transfected with wild-type GLUD1 or the R76K mutant. Western blot analysis of GLUD1 R76 methylation after immunopurified by Flag beads. H HEK 293T cells expressing Flag-tagged GLUD1 were treated with increasing concentrations of AdOx (0, 20 μM, 50 μM) for 24 h. Western blot analysis of R76 methylation after immunoprecipitation with Flag beads. I Both AGS and MFC cells were treated with 20 μM AdOx for 24 h. Western blot analysis of immunoprecipitated endogenous GLUD1 methylation.
