Figure 3. Translocation of 14-3-3γ from the cytoplasm to the inside of mitochondria.

(a) The mitochondrial localization of 14-3-3γ during MALM. The Ad-LacZ and Ad-Mieap-infected HCT116 cells were subjected to IF analysis on day 3 after the IR. The 14-3-3γ protein was stained with anti-14-3-3γ antibody (14-3-3γ:green). The Mieap protein was stained with anti-Mieap antibody (Mieap: red). The lysosomes were stained with anti-LAMP1 antibody (LAMP1: red). The mitochondria are indicated by the DsRed-Mito protein signal (Mito: red). The yellow area indicates overlapping between 14-3-3γ and mitochondria, Mieap, or lysosomes. A quantitative analysis of the yellow, red, or green area was performed using 300–400 cells. The average values of the yellow to red or green ratios (merged/mitochondria, 14-3-3γ, or lysosomes; yellow bar graph) are shown with error bars indicating 1 SD. P < 0.01 (*) was considered statistically significant. Scale bar = 20 μm. (b and c) The intramitochondrial localization of 14-3-3γ during MALM. The mitochondria were fractionated from the Ad-LacZ- and Ad-Mieap-infected HCT116 cells (a) or the control and Mieap-KD A549 cells (b) on day 3 after the IR. The mitochondrial fraction was subjected to a proteinase K protection assay. (−): no treatment, (+): proteinase K treatment, Mieap: Ad-Mieap-infected HCT116 cells, LacZ: Ad-LacZ-infected HCT116 cells, IR: ionizing radiation, Mitochondria: mitochondrial fraction, Cytosol: cytoplasmic fraction, and Whole: whole cell lysate. (b) C: HCT116 cells infected with Ad-LacZ, α: HCT116 cells infected with Ad-Mieap α, β: HCT116 cells infected with Ad-Mieap β. (c) C: A549 control cells, K: A549 Mieap-KD cells, α: endogenous Mieap α protein, and β: endogenous Mieap β protein. Full-length blots are presented in Supplementary Figure S8.