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. 2006 Sep 7;577(Pt 1):17–29. doi: 10.1113/jphysiol.2006.118299

Figure 1. Mitochondrial targeting of Kir6.2.

Figure 1

A, expression construct was generated by inserting the Kir6.2 gene in frame and downstream of the mitochondrial targeting sequence and green fluorescence protein (GFP) into the NotI restriction enzyme recognition site. B, confocal fluorescence imaging of HEK293 cells transfected with generated construct (green) and loaded with MFR, a mitochondrial-specific marker (red). GFP–Kir6.2 traffics to mitochondria, as seen by overlaying the green and red images (yellow). C, immunoblotting of cellular (cell), mitochondrial (mito) and microsomal (micro) fractions isolated from transfected HEK293 cells with Kir6.2 and cytochrome c oxidase (cyt-ox) (subunit I) antibody. Expression of GFP–Kir6.2 was detected in mitochondria of mito/GFP–Kir6.2 cells, but was not found in their plasma membrane. Mitochondrial expression was also confirmed in the cells transfected with the inactive form of Kir6.2 (mito/GFP–Kir6.2AAA). D, immunoblotting of mitochondrial fraction isolated from transfected HL-1 cells with the Kir6.2 antibody revealed a single band of ∼70 kDa in pCMV/mito/GFP–Kir6.2 transfected cells. Representative blots of three different experiments are shown.