Fig. 3: Gdh localization in brain and liver and Tet3 + Gdh interaction after ectopic co-expression in HEK293T cells.
From: Redirected nuclear glutamate dehydrogenase supplies Tet3 with α-ketoglutarate in neurons
a Immunofluorescence staining of Tet3 and Gdh in murine hippocampus compared to liver. Scale bar is 5 µm. b Western blot to determine Gdh content of different cellular compartments (fractionation from total murine brain) shows different echoforms of Gdh. CoxIV was used as a mitochondrial marker. c PLA in HEK293T after ectopic co-expression of GFP-Tet3 and Gdh-FLAG. GFP + Gdh-FLAG co-expressing cells were used as a negative control. In the merged image, GFP signal is shown in magenta, PLA signal in orange and nuclei (cyan) were stained with Hoechst. Scale bar is 50 µm. d Levels of hmdC, fdC, and cadC 10 h, 24 h, and 48 h after transfection of HEK293T (n = 3 biologically independent samples for each timepoint) with either GFP-Tet3 + Gdh-FLAG expressing plasmids (magenta) or GFP-Tet3 expression plasmid and an empty vector (cyan). Levels of mdC-oxidation products were normalized to the GFP signal of the cells. For each modification, each timepoint was compared individually. Two-sided t-test, correction for multiple comparisons using Holm-Sidak method, padj < 0.05 (*), <0.01 (**), <0.001 (***); exact p values and statistical details are provided in the Statistics and Reproducibility sub-section within the “Methods” section. Bars show mean, error bars show SD. LOD limit of detection e Levels of hmdC and fdC in the absence or presence of 20 µM of Gdh inhibitor R162 24 h after transfection of HEK293T (n = 2 biologically independent samples) with GFP-Tet3 and Gdh-FLAG expressing plasmids. a, c Images show Z-stacks. Source data are provided as a Source Data file.
