Extended Data Fig. 4: MISO forms inner mitochondrial membrane subdomains across a range of expression levels.
From: MISO regulates mitochondrial dynamics and mtDNA homeostasis by establishing membrane subdomains
a. Representative images showing the mitochondrial localization of MISO–Flag expressed in different cell types. Mitochondria were marked with HSP60. Scale bars: main panels 10 μm, magnified insets 2 μm. b. Immunoblot analysis of lysates from MISO-WT and MISO-KO expressing MISO–Flag cells by anti-MISO antibody. Arrowheads indicate the expected band, and asterisks denote non-specific bands. c. Representative images of MISO localization in U2OS cells expressing untagged MISO, detected with an anti-MISO antibody. Scale bars: main panel 10 μm, magnified insets 5 μm. d. A diagram of mMISO gene structure with 3×Flag tag knock-in element. e. Immunoblot analysis of endogenous mMISO expression across various mouse tissues. HSP60 and GAPDH were used as loading controls. f. A Scheme of the lentiviral cassette for the inducible expression of MISO–Flag. g. Western blot analysis of MISO–Flag expression in U2OS cells following induction with 10 ng/ml doxycycline. h. Representative images of MISO–Flag fluorescence intensity in mitochondrial filaments and subdomains across a range of expression levels. Scale bars: main panels 10 μm, magnified insets 2 μm. i. Representative images and corresponding colocalization analysis demonstrating MISO localization to the inner mitochondrial membrane. Scale basr: 1 μm. j. Cell fractionation assay of MISO–Flag stable expressing cells. WCL: whole cell lysate; Cyto: cytoplasm; Mito: mitochondria. k. Protease protection assay of mitochondria isolated from U2OS cells stably expressing MISO–Flag. l. Mitochondrial protein extraction was performed using sodium carbonate treatment. Total mitochondria (T) were separated into supernatant (S) and pellet (P) fractions.
