Fig. 4
From: Active and dynamic mitochondrial S-depalmitoylation revealed by targeted fluorescent probes
Overexpression of a mitochondrial-targeted APT selectively effects mitoDPP-2. a Immunostaining of HeLa cells overexpressing APT1 fused to a COX8 mitochondrial targeting sequence (COX8-APT1-myc) shows the fusion protein localized to mitochondria. b Structure of DPP-2, a previously reported, non-targeted cysteine deacylase probe. c HeLa cells transfected with COX8-APT1-myc or a catalytically inactive version, COX8-APT1(S119A)-myc, for 30 h, then loaded with 1 µM DPP-2 for 10 min, and analyzed by fluorescence microscopy. d Quantification of experiment described in (c) reveals that overexpressing a mitochondrial-targeted APT has no effect on the cytosolic APT signal as measured by DPP-2. e HeLa cells transfected with COX8-APT1-myc or a catalytically inactive version, COX8-APT1(S119A)-myc, for 30 h, then loaded with 500 nM mitoDPP-2 for 10 min, and analyzed by fluorescence microscopy. f Quantification of experiment described in (e) reveals that overexpressing a mitochondrial-targeted APT causes a significant enhancement on the mitochondrial APT signal as measured by mitoDPP-2. For all imaging, 20 µm scale bar shown. For plots, statistical analyses performed with a two-tailed Student′s t-test with unequal variance, ***P value < 5 × 10−7; n = 8 images from two biological replicates, error bars are ± s.e.m
