Luminescence reagent set

Human K562 leukemia cells were cultured in RPMI‐1640 medium (Fujifilm Wako Pure Chemical Co.) supplemented with 3% fetal bovine serum, penicillin, and streptomycin for 48 h, followed by incubation with various concentration of d‐Leu (0‐10 μmol/L) for 24 h at 37°C in 5% CO₂. The cells were collected analyzed by western blotting. Membranes were immunostained with primary antibodies against Nrt2 (1:2000 dilution, Cusabio Technology LLC.), HO‐1 (1:8000 dilution, Gene Tex Inc), SOD2 (1:4000 dilution, Cusabio LLC.), and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH, 1:8000 dilution, Fujifilm Wako Pure Chemical Co.), followed by incubation with anti‐rabbit IgG horseradish peroxidase (HRP)‐conjugated secondary antibody (Cell Signaling Technology Inc) for 1 hour at room temperature. Immunoreactive bands were visualized using a Luminescence Reagent Set (Wako Pure Chemical Industries, Ltd) and detected with Image Quant LAS500 (GE Healthcare Japan Com., Tokyo, Japan). The intensities of the detected bands were calculated using ImageJ software.

The collected ovaries (mice: n = 3 or 4/group) were homogenized in RIPA buffer containing 10 mmol/L sodium fluoride and 1 mmol/L phenylmethylsulfonyl fluoride as the phosphatase and protease inhibitors, respectively. The homogenates were centrifuged at 15,000 × g for 10 min at 4°C to obtain the supernatants. The supernatants were subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis and analyzed by Western blotting using the primary and secondary antibodies (anti-pACC [Ser79] Cat#11818; anti-ACC Cat#3676; anti-rabbit IgG HRP-linked antibody Cat#7074 from Cell Signaling Technology (Beverly, CA, USA); Anti-GAPDH Cat. 015-25473 from Wako Pure Chemical Corporation). Immunoreactive bands were visualized using a Luminescence Reagent Set (Wako Pure Chemical Corporation) and detected with Image Quant LAS500 (GE Healthcare Japan, Tokyo, Japan). The intensity of the detected bands was calculated using ImageJ software.