Hrp horseradish peroxidase conjugated secondary antibodies

For Western blot analysis, cells were lysed at 4°C in radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors cocktail (Sigma). Proteins were separated by SDS-PAGE and transferred onto 0.45-µm pore size polyvinylidene fluoride membranes (Immobilon-P PVDF membrane; Millipore). Polyvinylidene fluoride membranes were blocked with TBS-T (50 mM Tris, 150 mM NaCl, and 0.1% Tween-20) containing 5% (wt/vol) milk. Membranes were incubated with primary antibodies diluted from 1/500 to 1/1000, followed by TBS-T washes and incubation with HRP (horseradish peroxidase)−conjugated secondary antibodies (GE Healthcare). The signal was visualized by enhanced chemiluminescence with Luminata Forte Western HRP Substrate (Millipore) and the ImageQuant LAS 4000 imaging system. The following antibodies were used: anti-TFAM (Proteintech), anti-MT-CO1 (Millipore), anti-HIF1a and anti-mt-ND1 (Novus Biologicals), anti-P53 (Santa Cruz), anti-PGC1a (Thermo Scientific), anti-VDAC (Abcam), anti-Actin (Abcam), and anti–α-tubulin (Cell Signaling).

SDS-PAGE and immunoblotting were performed according to the method as described previously [7 (link),25 (link),26 (link)]. Total proteins (20–30 μg) were diluted in Laemmli buffer containing 2-mercaptoethanol, heated to 95°C for 5 min, separated on a 4-20% gradient Tris-glycine precast gel (Invitrogen) at 120 V for 1.5 h. Blots were incubated with primary antibodies specific for NP1 (1:500, BD Transduction Laboratories, Tamecula, CA, USA). HRP (horseradish peroxidase)-conjugated secondary antibodies (GE Healthcare, Piscataway, NJ, USA) were used at 1:10000 dilutions for 1 h at room temperature. The HRP reaction product was visualized using an ECL Western blotting detection kit (GE healthcare). Image films were scanned in gray scale (HP Scanjet G4010) at a high resolution as TIFF files. Immunoreactive protein bands corresponding to the correct molecular mass of target protein were quantified by drawing rectangle around the individual band and the intensity was measured by densitometry using NIH ImageJ software. Values were normalized to internal standard actin, which also serve as a loading control, to make relative comparisons.

SDS/PAGE and immunoblotting were performed according to the method as described previously [10 (link),26 (link)]. Total proteins (20–30 μg) were diluted in Laemmli buffer containing 2-mercaptoethanol, heated to 95°C for 5 min and separated on a 4-20% gradient Tris-glycine precast gel (Invitrogen) at 120 V for 1.5 h. Blots were incubated with primary antibodies for PGC 1α (1:1000; Abcam), TFAM (1:1000; Sigma, St Louis, MO, USA), HSP60 (1:1000; Cell Signaling, Beverly, MA, USA), COX IV (1:1000; Cell Signaling) and actin (1:5000, mouse monoclonal anti b-actin antibody; Sigma.). HRP (horseradish peroxidase)-conjugated secondary antibodies (GE Healthcare, Piscataway, NJ, U.S.A.) were used at 1:10000 dilutions for 1 h at room temperature. The HRP reaction product was visualized using an ECL Western blotting detection kit (GE healthcare). Image films were scanned in gray scale (HP Scanjet G4010) at a high resolution as TIFF files. Immunoreactive bands corresponding to the correct molecular mass of target protein were quantified by drawing rectangle around the individual band and the intensity was measured by densitometry using NIH ImageJ software. Values were normalized to internal standard actin, which also serve as a loading control, to make relative comparisons.