Hrp conjugated anti rabbit or anti mouse igg antibody

Ten male flies were used for each condition. The antibodies used in Western blot analysis were as follows: anti-4HNE (1:1000, JalCA); anti-ATP5A (1:100,000, Abcam); and anti-tubulin (1:10,000, Sigma). HRP-conjugated anti-rabbit or anti-mouse IgG antibodies (Cell Signaling Technology) were used as secondary antibodies. Blots were visualized using Chemi-Lumi One Super reagent (Nacali Tesque Inc.). To quantify 4-HNE and ATP5A levels, staining intensity of each lane was measured using ImageJ (http://imagej.nih.gov/ij/). Measured area is indicated in the box.

In brief, the cells were homogenized in mammalian protein extraction buffer (GE Healthcare) with a protease inhibitor cocktail (MCE) after the various treatments. Protein samples were electrophoresed on a 10% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane (Bio-Rad). Strips from the membrane were blocked with 5% non-fat milk in Tris-buffered saline, pH 7.4, containing 0.1% Tween (TBS-Tween). The strips were incubated with primary antibodies against phosphorylated-AKT (p-AKT)/AKT and p62 (Santa Cruz), integrin β1, β3, p-FAK/FAK, p-paxillin/paxillin (BD Biosciences), p-mTOR, mTOR, LC3β, and GAPDH (Cell Signaling) at 4°C overnight. After being washed, the strips were incubated with HRP-conjugated anti-rabbit or anti-mouse IgG antibodies diluted 1:5,000 (Cell Signaling). Next, the blots were incubated in ECL reagent for signal development (Bio-Rad). The densities of the bands on the nitrocellulose membrane were captured by SageCapture Microsoft Basic Application software and quantified by densitometry using Gel Pro 3.1 (Media Cybernetics), with the density of the control sample set to 100% and the density of each test sample expressed relative to the expression of the internal control. The phosphorylated proteins were normalized to total protein first.

Ten male flies aged 3–5 days were used for each condition. The bodies were dissected out in Schneider’s Drosophila medium and homogenized in sample buffer (Nacali Tesque Inc.). For insulin treatment, the bodies were incubated with or without 5 μM recombinant human insulin (Sigma, I2643) for 1 h at 25°C before being homogenized in sample buffer. Rabbit anti-pAKT (1:1000, Cell signaling); rabbit anti-AKT (1:1000, Cell signaling); and mouse anti-tubulin (1:10,000, Sigma) were used for primary antibodies, and HRP-conjugated anti-rabbit or anti-mouse IgG antibodies (Cell Signaling Technology) were used for secondary antibodies. Blots were visualized using the Chemi-Lumi One Super reagent (Nacali Tesque Inc.).