Gel pro analyzer 4

Total proteins of A7r5 cells treated with ox-LDL and β-GP or ox-LDL, CML, and β-GP were extracted by centrifugation in RIPA lysis buffer. The protein samples were loaded onto SDS-polyacrylamide gel and then transferred onto polyvinylidene difluoride (PVDF) membranes using a semidry method. The membranes were incubated with the primary antibodies anti-BMP-2, anti-Runx2, anti-ALP, and anti-GAPDH overnight at 4°C and then with HRP-conjugated secondary antibodies for 1 h at room temperature. The immunoreactive bands were visualized with an ECL kit (Thermo Fisher Scientific, Rockford, USA), and images were obtained using Gel-Pro Analyzer 4 software (Media Cybernetics, USA).

To determine the effect of the studied amino acid substitutions on the stage of enzyme binding to a DNA substrate containing a gap, the electrophoretic mobility shift assay was used. The reaction was carried out in a buffer composed of 50 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM Na₂EDTA, 5 mM MgCl₂, 1 mM DTT, and 7% glycerol. The recombinant enzymes were serially diluted; for the Polβ L19P polymorphic variant, the reaction was carried out in the concentration range from 57 nM to 7.3 μM, and for the G66R polymorphic variant, from 55.5 nM to 7.1 μM. The samples were incubated for 15 min at room temperature and applied to a nondenaturing 10% polyacrylamide gel (PAAG; the ratio of acrylamide to N,N′-methylenebisacrylamide was 75:1).
To determine the dissociation constant, the resultant gel was visualized in a VersaDoc gel-documenting system (Bio-Rad Laboratories, Hercules, CA, USA). The results were processed using Gel-Pro Analyzer 4 software (Media Cybernetics, Rockville, MD, USA). Dissociation constant K_d for each enzyme–DNA complex was computed in OriginPro 8 software via the following equation:
where h is the Hill coefficient, Fu is the correction for background illumination, and Fb is the maximum intensity of the complex.

Brain tissues were harvested and homogenized on ice before centrifugation at 13,000×g at 4 °C for 5 min. The supernatants were collected and saved at −20 °C. Total protein concentration was measured with the bicinchoninic acid method (Beyotime Biotechnology, Co., Ltd., Jiangsu, China). The samples were separated using 12% gel electrophoresis (90 V for 25 min, then 120 V for 1 h) and then transferred to a nitrocellulose membrane at 200 mA for 1 h. The blots were blocked in 5% milk Tris-buffered saline and Tween-20 (TBST) buffer for 1.5 h at 25 °C, and then incubated with a monoclonal antibody against caspase-3 (1:1000; Cell Signaling Technology, Inc. #9662, Danvers, MA, USA) or β-actin (1:1000; Cell Signaling Technology) in 5% milk TBST buffer at 4 °C overnight. After washing in TBST, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:6000 dilution for β-actin and caspase-3, Wanleibio Co., Ltd., Shenyang, China) for 2 h at RT. Following the ECL reaction, the membranes were subjected to autoradiography and films were scanned using Tanon Imager 5200 software v2.03 (Tanon Co., Ltd., Shanghai, China). Western blot band intensity was quantified by the mean pixel intensity using Gel-Pro Analyzer 4 software (Media Cybernetics Inc., Bethesda, MD, USA).