Figure 2 | Scientific Reports

Figure 2

From: Protein N-myristoylation plays a critical role in the mitochondrial localization of human mitochondrial complex I accessory subunit NDUFB7

Figure 2

DMAC1, HCCS, NDUFB7, and PLGRKT are N-myristoylated mitochondrial proteins. Seven proteins (CLN3, DMAC1, HCCS, MARC1, NDUFB7, NOL3, and PLGRKT) were selected from proteins in which protein N-myristoylation was detected in X(N10)-tGelsolin-FLAG, and their protein N-myristoylation and intracellular localization were evaluated using C-terminally FLAG-tagged full-length proteins. (A) Analysis of the protein N-myristoylation of CLN3, DMAC1, HCCS, MARC1, NDUFB7, NOL3, and PLGRKT expressed in transfected COS-1 cells by metabolic labeling. cDNAs coding C-terminally FLAG-tagged full-length proteins were transfected into COS-1 cells and cells were then labeled with a myristic acid analog. The expression of proteins was evaluated by Western blotting using an anti-FLAG antibody (left panel). Protein N-myristoylation was evaluated by metabolic labeling followed by click chemistry, as described in the Methods (right panel). Arrows indicate the position of the expressed proteins. The images obtained by Western blotting or metabolic labeling were cropped. Uncropped full-length raw image data are shown in Supplementary Figure S2. (B) Analysis of the intracellular localization of CLN3, DMAC1, HCCS, MARC1, NDUFB7, NOL3, and PLGRKT by an immunofluorescence microscopic analysis. The intracellular localization of seven proteins was assessed by an immunofluorescence analysis of COS-1 cells transfected with cDNA coding C-terminally FLAG-tagged full-length proteins using an anti-FLAG antibody. MIC19-FLAG was used as a control mitochondrial N-myristoylated protein. Hoechst and MitoTracker Red were used as organelle markers for the nucleus and mitochondria, respectively. Experiments were repeated 3 times and similar results were obtained. Representative data are shown. Abbreviation used: PC, phase contrast image. The results of the line profile analysis are shown. In the line profile analysis, corresponding line-scan graphs of the relative fluorescence intensities of green anti-FLAG fluorescence (green line) and red MitoTracker red fluorescence (red line) along the white line indicated in the merged images are shown.

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