iJC-TCRβ localizes to the mitochondrial outer membrane and induces cytochrome c release from mitochondria. (A) COS-7 cells were transfected with either EGFP-iJC (i,ii) or EGFP-VDJC-KKKNS (iii), and 24 hours later they were fixed using the high-powered fields (HPF) method for TEM. The cells were either left unstained (i) or stained by anti-GFP (ii,iii) followed by goat anti–rabbit antibodies coupled to 10-nm gold particles. Arrowheads indicate the thickening of the mitochondrial membrane in transfected cells, which is shown at higher magnification in the top left insert. The insert at the bottom right corner of Ai shows a normal double mitochondrial membrane negatively stained. (iv) 293T cells were transfected with EGFP-iJC, and 24 hours later mitochondria were isolated. Mitochondria were then either treated with proteinase K or left untreated. Proteins were extracted, separated by SDS-PAGE, and blotted with anti-GFP stripped and reblotted with anti-Hsp60. (B) COS-7 cells were transfected with either EGFP-iJC (i) or EGFP(-Ig)C (iii,iv) and 24 hours later were fixed using the HPF method. The cells were than stained by either anti–cytochrome c (i,iii) or anti-GFP antibodies (iv) followed by goat anti–rabbit IgG coupled to 10-nm gold particles. Each of the images in subpanels i, iii, and iv shows a representative mitochondria. The amount of intramitochondrial gold particles in a square U of mitochondria was compared between 13 cells that were transfected with either EGFP-iJC or EGFP(-Ig)C, and the results are represented as a graph (Biib). Bar, 0.5 μM.