Rearranged TCRβ, harboring a complete V region and lacking the leader sequence, localizes to the mitochondrion and induces apoptosis. (A) Schematic presentation of the different constructs of a rearranged TCRβ (B) COS-7 cells were transfected with the indicated VDJC-TCRβ constructs and 24 hours later were either incubated with MTR for 30 minutes or stained with either anti-KDEL or anti-Hsp60 followed by anti–mouse Cy3 antibodies. The left column shows EGFP images, whereas the middle column shows the MTR or KDEL signal (i) or anti-Hsp60 (ii). The right column is the merge of the EGFP and MTR, KDEL or Hsp60 signal. Fluorescent images were taken by confocal microscopy (i) or fluorescence microscopy (100× oil-immersion objectives) (ii). Bar, 10 μM. (C) 293 cells were transfected with the indicated constructs and 48 hours after transfection were subjected to a flow cytometry cell-cycle analysis. Cells transfected with empty vector are pEGFP-F (Civ). (D) Schematic presentation of Leader-Flag-VDJC. (E) 293 cells were transfected with the indicated constructs. At 48 hours after transfection cells were subjected to a flow cytometric cell-cycle analysis. Cells transfected with an empty pCDNA3 vector are shown (Ev) as indicated. (F) The most COOH-terminal residues of mouse TCRα, β1, and β2 are shown. The positively charged residues adjacent to the putative TMD are marked in red (i). COS-7 cells were transfected with ECFP-TCRα and 24 hours later were stained with anti-Hsp60 antibody followed by a Cy3-conjugated secondary antibody (red) (ii). Fluorescent images were taken by fluorescence microscopy (100× oil-immersion objectives). Bar, 10 μM.