Rat femora were subjected to cell lysis to extract proteins. The concentration of total protein was determined using a BCA protein assay kit. Proteins (30 µg) were separated by 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with a buffer containing 0.05% Tween-20 and 5% defatted milk and reacted sequentially with primary antibodies against GAPDH and N1ICD (1:1,000) for 10 h at 4°C and HRP-conjugated anti-rabbit secondary antibody (1:3,000) for 1 h at 25°C. The membranes were washed and rinsed with enhanced chemiluminescence (ECL) detection reagents (EMD Millipore, Billerica, MA, USA). The band images were photographed using ECL. Immunoreactive bands were visualized using ECL substrates and an X-ray film processor. Protein expression was calculated using Quantity One® software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA).
Quantity one software
Manufactured by Media Cybernetics
Sourced in United States
Quantity One software is a data analysis and image processing tool. It allows users to capture, analyze, and quantify digital images.
Automatically generated - may contain errors
Lab products found in correlation
Ecl detection reagent, by Merck Group (1 mentions)
Ab7671, by Abcam (1 mentions)
Ab3583, by Abcam (1 mentions)
Ab133504, by Abcam (1 mentions)
Ab4051, by Abcam (1 mentions)
Ab59348, by Abcam (1 mentions)
Ab108400, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
G box system, by Syngene (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Complete mini, by Roche (1 mentions)
6 protocols using quantity one software
1
Rat Femoral Protein Extraction and Quantification
2
Western Blot Analysis of Cardiac Proteins
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Expression levels of leptin, caspase 3, Bax, Bcl‐2, NKA, calpain, sodium‐calcium exchanger 1, and β‐actin proteins were determined by Western blot, as described previously.17 The bands of target proteins and internal reference β‐actin were scanned and analyzed by Quantity One software (Media Cybernetics Inc). The results are shown as the ratio of integrated optical density of the target proteins/the internal control and statistically analyzed. The primary antibodies used were anti–β‐actin antibody (JC‐PA002; 1:1000; Jingcai Biology, China), anti–caspase 3 antibody (ab4051; 1:500; Abcam), anti‐Bax antibody (1:1000; Abcam), anti‐leptin antibody (ab3583; 1:1000; Abcam), anti–Bcl‐2 antibody (ab59348; 1:1000; Abcam), anti–cytochrome C antibody (ab133504; 1:1000; Abcam), anti–calpain 1 antibody (ab108400; 1:5000; Abcam), and anti‐α1 NKA antibody (ab7671; 1:1000; Abcam).
3
Western Blot Analysis of PC12 Cells
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PC12 cells were subjected to lysis with RIPA buffer after treatment. Protein concentration was determined by a BCA kit, and 100 μg of total protein was separated by 10%SDS-PAGE electrophoresis. The proteins were then transferred to a PVDF membrane. The membrane was blocked with 5% milk for 1 h at room temperature, and the primary antibodies were incubated at 4°C overnight. After washing, the secondary antibodies were added and incubated at room temperature for 1 h. Chemiluminescence was used for color development, and image analysis was performed with Quantity One software (Media Cybernetics, Inc., Rockville, MD, USA).
4
Quantifying Oxidative Stress in HUVECs
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HUVECs were seeded at 1.0 × 106 cells/well in 6-well plates and allowed to grow attached to the wall overnight. Cells were then incubated continuously with serum-free medium for 12 h, after which HUVECs were treated with 0 and 250µM of H2O2 for 24 h. Finally, the medium was collected and centrifuged. The BCA method was used to determine the protein concentration in the culture medium, which was used to adjust the amount of protein samples. APMA, 4-Aminophenylmercuric acetate, was selected as a positive control. The gel was subjected to two shaking elution for 20 min each, followed by two rinses of 20 min each with the rinse solution (Triton X-100 free in the eluent). Then it was put in the incubation solution on the shaker for 42 h. After 3 h of staining, the gels were dyed blue and then treated with 10% decolorizing solution for 0.5 and 1 h and 5% decolorizing solution for 2 h, respectively. Ultimately, the locations where the white and translucent bands appear were observed. All images were analyzed by Quantity One software (Media Cybernetics, MD).
5
Western Blot Analysis of Endothelin-1
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PVECs were prewashed and placed into a solution mixed with radioimmunoprecipitation assay buffer and protease inhibitors. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Beyotime, China). Equivalent amounts of protein were separated in 10% polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore). Membranes were blocked in 5% nonfat milk and incubated with primary antibodies, including anti-endothelial-1 (5 µg/mL; Abcam, United Kingdom) and anti-βactin (0.1 µg/mL, Sigma-Aldrich), overnight. After incubation with corresponding secondary antibodies at ambient temperature, protein bands were coated with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and visualized using a G:BOX system (Syngene). Quantification of bands was carried out using Quantity One software (Media Cybernetics) and normalized to β-actin.
6
Quantifying HIF-1α Expression in Muscle Tissue
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The excised quadriceps femoris muscles were homogenized in ice-cold buffer (8 M urea,
1/10 v/v glycerol, 1/20 v/v 20% SDS, 1/200 v/v 1 M dithiothreitol, 1/100 v/v 0.5 M
Tris, adjusted to pH 6.8) containing a protease inhibitor cocktail (Complete Mini,
Roche, China), prior to PAGE and transfer onto polyvinylidene fluoride membranes
(Wuhan Boster Co.). The membranes were incubated with either mouse monoclonal
anti-HIF-1α antibody (1:500, Wuhan Boster Co.) or rabbit polyclonal anti-β-actin
antibody (1:2,000, Wuhan Boster Co.). Bound antibody was visualized with
species-specific horseradish peroxidase-conjugated secondary antibody and a
chemiluminescence system (Wuhan Boster Co.). Signals were quantified using a Gel Doc
2000 scanner with the Quantity One software (Media Cybernetics Co.). The densities of
bands were normalized to β-actin.
1/10 v/v glycerol, 1/20 v/v 20% SDS, 1/200 v/v 1 M dithiothreitol, 1/100 v/v 0.5 M
Tris, adjusted to pH 6.8) containing a protease inhibitor cocktail (Complete Mini,
Roche, China), prior to PAGE and transfer onto polyvinylidene fluoride membranes
(Wuhan Boster Co.). The membranes were incubated with either mouse monoclonal
anti-HIF-1α antibody (1:500, Wuhan Boster Co.) or rabbit polyclonal anti-β-actin
antibody (1:2,000, Wuhan Boster Co.). Bound antibody was visualized with
species-specific horseradish peroxidase-conjugated secondary antibody and a
chemiluminescence system (Wuhan Boster Co.). Signals were quantified using a Gel Doc
2000 scanner with the Quantity One software (Media Cybernetics Co.). The densities of
bands were normalized to β-actin.
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