iJC-TCRβ localizes to mitochondria in both T and non-T cells. (A) Schematic representation of EGFP-iJC and different modified and truncated versions of this molecule. (B) Different T cells [S49.1 (i), SCID.adh (ii)], and thymocytes from either wild-type (iii) or TCRβ-deficient mice (iv) were transfected with EGFP-iJC (green) and 5 hours later were stained with anti-Hsp60 antibody followed by a Cy3-conjugated secondary antibody (red) and with Hoechst for nuclear staining (blue). The marked area is magnified in the inset. Bar, 1 μM. (C) COS-7 cells were transfected with EGFP-iJC (green) and 24 hours later were incubated with MTR (red) for 30 minutes (i). COS-7 cells were transfected with Flag-iJC, 24 hours later the cells were incubated with MTR (red) for 30 minutes and stained by either anti-Flag (ii) or anti-TCR (iii) antibodies followed by Alexa 488–conjugated secondary antibody (green). COS-7 cells were transfected with EGFP-iJC (green) and 24 hours later stained with anti-Hsp60 antibody followed by a Cy3-conjugated secondary antibody (red) (iv). (D) The cell line 293T was transfected with EGFP or EGFP-iJC-TCRβ. Forty-eight hours after transfection cell fractionation was preformed, followed by Western analysis using anti-GFP antibody. Fluorescent images (B,Civ) were taken by fluorescence microscopy (100× oil-immersion objectives) and fluorescent images (Ci-iii) were taken by confocal microscopy. TCR mitochondrial localization was found in all of a total of 100 cells examined in each experimental group, within this figure and the figures below. Constructs are described in Figure 1A. Bars, 5 μM (B), 10 μM (C).