Fig. 5: GRAMD1C interacts with the mitochondria through the GRAM domain.

a Overview of the EGFP-tagged GRAMD1C constructs used. TM = Transmembrane domain. b U2OS cells stably expressing GRAMD1C-EGFP were stained with Mitotracker Red and subjected to live cell confocal microscopy. The graphs depict the pixel intensity along the white lines drawn in the insets (1 and 2). Scale bar =  20 µm, inset = 2 µm. n = 3 independent experiments, >5 cells each experiment. c U2OS cells stably expressing EGFP-GRAM domain were stained with Mitotracker Red and subjected to live cell confocal microscopy. The insets (1 and 2) represent snapshots showing the recruitment of EGFP-GRAM to the mitochondria. Scale Bar = 10 µm. n = 3 independent experiments, >5 cells each experiment. d Mitochondria were isolated using percoll density centrifugation from U2OS cells expressing EGFP or EGFP-GRAM and subjected to western blot analysis for the indicated proteins. TCL total cell lysate. n = 3 independent experiments e GRAMD1C-EGFP was immunopurified from stably transfected U2OS cells, and copurified proteins were identified using mass-spectrometry analysis. The interactome of GRAMD1C-EGFP was compared to the interactome of EGFP. Significant hits (P < 0.05) are colored blue or brown (mitochondrial). f EGFP, EGFP-GRAM, GRAMD1C-EGFP, and ΔGRAM-EGFP were immunoprecipitated from stably transfected U2OS cells and the precipitate subjected to western immunoblot analysis for the indicated proteins. n = 3 independent experiments. Source data are provided as a Source Data file.