Gradient tris glycine precast gel

Adult mice of indicated genotypes were killed, and retinas were dissected and lysed with lysis buffer (Cell Signaling Technology) containing 0.5 mm phenylmethylsulfonyl fluoride (Sigma-Aldrich). Protein concentration was determined by BCA protein assay (Thermo Fisher Scientific). Samples (25 μg) were separated by SDS-PAGE in 4-20% gradient Tris-glycine precast gels (Invitrogen) and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was incubated for 1 h in blocking solution containing 5% nonfat milk powder and 0.1% Tween-20, pH 7.6. This was followed by overnight incubation at 4°C in the blocking buffer containing rabbit primary antibodies against KLF4 (1:50; Ab72543, Abcam). Subsequently, the labeled proteins were visualized by incubation with a horseradish peroxidase (HRP)-conjugated anti-goat or rabbit IgG (1:2000; Santa Cruz Biotechnology) followed by development with a chemiluminescence substrate for HRP (Thermo Fisher Scientific). The images of Western blots were captured by GE ImageQuant. Relative band intensities were analyzed using ImageJ software and normalized to GAPDH.

Experimental rats were sacrificed 12 hours after ROSC. Then, the rat hearts were harvested and the left ventricular myocardial tissue was collected, homogenized, and lysed with lysis buffer (Cell Signaling Technology, Boston, MA, USA) containing 0.5 mM of phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was determined by BCA protein assay (Thermo Fisher Scientific, Grand Island, NY, USA). Samples of 25 μg protein were fractionated by SDS-PAGE in 4–20% gradient Tris-glycine precast gels (Invitrogen) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated for 1 hour in blocking solution containing 5% nonfat milk powder and 0.1% Tween-20, pH 7.6. This was followed by an overnight incubation at 4°C in blocking solution containing rabbit primary antibodies against KEAP1 (D6B12, Cell Signaling Technology, Boston, MA, USA). Subsequently, the labeled proteins were visualized by incubation with a horseradish peroxidase- (HRP-) conjugated anti-goat or rabbit IgG (1 : 2000; Santa Cruz Biotechnology) followed by development with a chemiluminescence substrate for HRP (Thermo Fisher Scientific). The images of Western blots were captured by GE imageQuant.

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.