Extended Data Fig. 4: Proximity-based fluorescent labelling by TNAP–APEX2 and trypsin protection assay on mitochondria from BAT.
From: Mitochondrial TNAP controls thermogenesis by hydrolysis of phosphocreatine
a, Confocal fluorescence microscopic images of immortalized brown adipocytes showing co-localization of the GFP signal from 3XHA-EGFP-OMP25 construct (mGFP, channel: 488 nm) with different mitochondria markers. Endogenous antibodies, OxPhos (upper red, channel: 561 nm) and HSP60 (lower red, channel: 640 nm), were used to visualize mitochondria. The insets show a magnified region of the image outlined by the dotted box. Scale bars, 5 μm. b, Illustration of how APEX2 reports subcellular localization of TNAP by its peroxidase activity. X indicates either Alexa Fluor 647-conjugated tyramide (for confocal microscopy) or 3,3′-diaminobenzidine (for TEM studies). c, Confocal fluorescence analysis of immortalized brown adipocytes (top) and hepatocytes (bottom) ectopically expressing a TNAP–APEX2 construct. Cells were fixed and treated with Alexa Fluor 647–tyramide/H2O2 for proximity-based fluorescent labelling facilitated by the peroxidase activity of APEX2. Stably expressed 3XHA-EGFP-OMP25 was used as mitochondria reporter. Scale bars, 10 μm. d, Western blot analysis of the protease protection assay on mitochondria derived from BAT of cold-acclimated mice. TOMM20, GPD2 (glycerol-3-phosphate dehydrogenase), Cyt C (cytochrome c) and CS (citrate synthase) are shown as markers of outer mitochondrial membrane (OMM), intermembrane space (IMS) and mitochondrial matrix. Blots were processed in parallel with samples derived from the same experiment. e, Relative protein abundances in trypsin-digested mitochondria derived from band intensities of intact protein quantified from d; n = 2 technical replicates. Data are presented as mean ± s.e.m.
