Figure 2

Internalization of TAT-ORF into isolated mitochondria and cells of F528 patients. (A, B) Internalization of TAT-ORF into mitochondria isolated from cells of F528 patients. Isolated mitochondria were added with TAT-ORF (0.11 µg/µL; final concentration) for 2 h, washed and submitted to Western blot analysis by using anti-His (A) (1:30,000 dilution) or anti-ORF (B) (1:10,000 dilution) antibodies. (C, D) Internalization of TAT-ORF into cells from F528 patients. F528 cells were treated with TAT-ORF (0.02 µg/µL; final concentration) for various time periods. The cells were washed and mitochondria were isolated and submitted to Western blot analysis using anti-ORF (C1 1:50,000 dilution), anti-E1α (C2; 1:1,000 dilution) or anti-His (C3; 1:10,000 dilution) antibodies. (D) Repeating the experiment with a higher concentration of the fusion protein added to the F528 cells (0.1 µg/µL, final concentration) and loading twice the amount of purified mitochondria (25 µg protein per lane), from both F528 treated cells and normal fibroblast. Proteins were identified by using anti-ORF antibodies. Arrows indicate the TAT-ORF fusion protein (A–C1, 3; D, right image), the E1α (C2) or the endogenous C6ORF66 protein (D, left image). (E) After internalization of FITC-labeled TAT-ORF into F528 cells for 1 h (E1), 2 h (E2) and 3 h (E3), by using confocal microscopy. Magnification 60×.