Blocking one

To detect aggregates of GAPDH in insoluble and mitochondrial fractions, each fraction was mixed with low SDS sample buffer (final concentration: 62.5 mm Tris-HCl, pH 6.8, 0.5% SDS, 10% glycerol, and 0.002% bromphenol blue) and then heated at 100 °C for 5 min. These samples were separated by 5–20% non-reducing SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad). The membranes were incubated for 1 h with Blocking One (Nacalai Tesque) to block nonspecific binding. The membrane was then incubated for 2 h at room temperature with an anti-GAPDH monoclonal antibody (1:300) in 10% Blocking One-PBST (0.05% Tween 20 and 0.02% NaN₃ in PBS) followed by incubation for 1 h at room temperature with a peroxidase-conjugated affinity-purified secondary antibody (Invitrogen). The signals were detected using both SuperSignal West Pico chemiluminescent substrate (GE Healthcare) and LAS3000 (Fujifilm, Tokyo). To assess the purity of mitochondria isolated from cells, Western blotting was performed using an anti-CIV monoclonal antibody (1:1000, a mitochondrial marker), an anti-histone H2B polyclonal antibody (1:5000, a nuclear marker), or an anti-TPI polyclonal antibody (1:1000, a cytosolic marker); membranes were also blotted with an anti-GAPDH antibody.

The frozen tissue sections were fixed with a solution containing a mixture of acetone and methanol in equal amounts. After air-drying, the dyed sample was surrounded by a water repellent pen (Dako). Then, permeabilization was performed thrice using 0.05% PBS with Tween 20 (PBST) for 10 min and then blocked at room temperature for 1 h using Blocking One (Nacalai Tesque). Thereafter, the primary antibody solution was diluted to an appropriate concentration with Blocking One and then stored at 4 °C overnight. After the reaction, the slide was washed thrice with PBST for 10 min. The secondary antibody solution was appropriately diluted with Blocking One (1: 500) and stored at room temperature for 1 h. After washing with PBS for 10 min, we added a mixture of 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Invitrogen) and Apaci mounting agent (Wako) dropwise at 1:1000, covered with a glass slide (Matsunami Glass) and then sealed. Antibodies were as follows: CD26 (BD Bioscience 559639), CD31 (BD Bioscience 550300), CK19 (Progen Biotechnik GmbH, 61029), SE-1 (Immuno-Biological Laboratories, 10078) and HNF4 alpha (H-1) (Santa Cruz, sc-374229).