FIGURE 6.

PGAM5 is required for localization of Nrf2-INrf2-PGAM5-Bcl-xL complex to the mitochondria. A, imunocytochemistry for localization of Nrf2-INrf2-PGAM5-Bcl-xL complex to the mitochondria. Hepa-1 cells were grown on coverslips, fixed, permeabilized, washed, and first incubated with a 1:500 dilution of anti-cytochrome c sheep antibody for 12 h. After washing, cells were incubated with 1:1000 dilution of anti-Nrf2 rabbit, anti-INrf2 goat, anti-Bcl-xL mouse, and anti-PGAM5 rabbit antibody for 12 h separately. After washing with PBS, cells were incubated with anti-sheep FITC-conjugated second antibody or Alexa Fluor-594-conjugated anti-goat, anti-rabbit, and anti-mouse second antibody for 1 h. After immunostaining, cells were observed under a Nikon fluorescence microscope, and photographs were captured. B, Hepa-1 cells were transfected with 100 nm control or PGAM5 siRNA, and cytosolic and mitochondrial fractions were prepared. Sixty-μg extracts were immunoblotted with anti-PGAM5L, anti-Bcl-xL, anti-INrf2, anti-Nrf2, anti-lactate dehydrogenase (LDH), and anti-CoxIV antibodies.