Extended Data Fig. 3: Mitochondrial localization of endogenous TNAP in BAT and non-thermogenic fat cells.
From: Mitochondrial TNAP controls thermogenesis by hydrolysis of phosphocreatine
a, Confocal fluorescence images showing subcellular localization of endogenous TNAP in brown adipocytes (top and middle panels) and hepatocytes (bottom panels). Primary brown preadipocytes were prepared from Alplfl/fl mice, transduced with either AdGFP (WT) or AdCre (Alpl KO) on day 4 of differentiation, and fixed for imaging on day 8. Arrows denote selected peri-nuclear areas of TNAP signal that co-localize with mitochondria signal. Antibodies for TNAP (red) and HSP60 (green) were used to visualize TNAP and mitochondria. Scale bar, 5 μm. b, Pearson’s correlation coefficient (PCC) analysis showing the extent of co-localization of TNAP with mitochondria in indicated cell types; n = 10 cells per group; data are presented as mean ± s.e.m.; statistical significance was calculated by one-way ANOVA with Bonferroni’s multiple comparisons test. c, Western-blot analysis on TNAP in wild-type versus knockout cells. Vinculin (VCL) blot was used as a sample preparation control. d, Confocal fluorescence microscopic images showing subcellular localization of endogenous TNAP in different cell types. Anti-TNAP and anti-HSP60 were used to visualize TNAP and mitochondria, respectively. Scale bars, 5 μm. e, Western-blot analysis on TNAP and mitochondrial markers in mitochondrial preparations from BAT of cold-acclimated, wild-type vs adipo-Alpl knockout mice. Blots were processed in parallel with samples derived from the same experiment. f, Western-blot analysis of the insoluble fraction of mitochondria extract treated with phosphatidylinositol-specific phospholipase-C (PI-PLC), followed by ultracentrifugation, showing that PLC treatment releases TNAP from membranes. P, pellet; S, supernatant. Mitochondrial preparation was fragmented by sonication before treatment. Blots were processed in parallel with samples derived from the same experiment.
