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. Author manuscript; available in PMC: 2021 Feb 3.
Published in final edited form as: Nat Cell Biol. 2020 Aug 3;22(8):973–985. doi: 10.1038/s41556-020-0549-1

Extended Data Fig. 2. Interactions and localization analyses of IRGM with MiT/TFE family of transcriptional regulators.

Extended Data Fig. 2

a, Confocal microscopy analysis of co-localization between GFP-IRGM and endogenous TFEB. Scale bar 5 μm, (n=3 biologically independent experiments). b, A screenshot from NCBI showing domain of unknown function (DUF3371) in TFEB. c,d, HCM images and quantifications to analyze the effect of complementation of IRGM KD with GFP-IRGM WT or GFP-IRGM S47N on nuclear translocation of TFEB. Data, means ± SEM (n=3 biologically independent experiments) ANOVA, Tukey’s post hoc test; HCM, >500 cells counted per well; minimum number of valid wells 9, 3 independent experiments. Masks; white: algorithm-defined cell boundaries and computer-identified GFP positive cells; blue outline: computer-identified nuclear stain; yellow outline: computer-identified colocalization between TFEB and Hoechst-33342 nuclear stain). Scale bar 10 μm. The masks in gray scale panels are cloned from the merged images. Inset: western blot showing GFP-IRGM expression IRGM KD cells. e, Co-IP analysis of interactions between GFP-MiTF (H isoform) and FLAG-IRGM in 293T cells, (n=3 biologically independent experiments). f, Co-IP analysis of interactions between GFP-TFE3 and FLAG-IRGM in 293T cells, (n=3 biologically independent experiments). g,h, Co-IP analysis of interactions between GFP-IRGM WT or GFP-IRGM S47N with MiTF in 293T cells. Data, means ± SEM of normalized intensities (n=3 biologically independent experiments) paired t-test. Uncropped blots for panels e, f and g are provided in Unprocessed Blots Data Extended Fig. 2 and numerical source data for panels c and h are provided in Statistical Source Data Extended Fig. 2.