Ab2302

Western blot analysis was used to analyze changes in the expression of Cx43 protein. Briefly, proteins from each group of BAs or SMCs were subjected to SDS-PAGE and then transferred to nitrocellulose membranes, which were blocked for 2 h in Tris-buffered saline Tween (TBST) containing 5% milk powder at room temperature. The anti-Cx43 antibody (MAB3067; Sigma, diluted 1:1000) and anti-GAPDH antibody (AB2302; Sigma, diluted 1:1000) were used to the membrane in saturation buffer. After extensively washing 5 times in TBST for 10 min each, the membranes were probed for 2 h with horseradish peroxide-conjugated goat anti-rabbit secondary antibody (diluted 1:2000 in saturation buffer). The durations of exposure to hyperfilm and incubation in ECL detection reagents were maintained consistent for all conditions. The intensity of the bands after Western blotting was determined by laser scanning of the films, followed by quantitative densitometric analysis using Kodak Digital Science 1D 2.0 Image software, and the protein expression levels of Cx43 were normalized to GAPDH.

Tissues were homogenized in lysis buffer containing 50 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton-X 100 and protease inhibitors (Roche). Cell lysates were centrifuged at 13,000 g for 15 min at 4 °C. Protein quantification was performed with a BCA kit (Pierce). Supernatant was mixed with 5 × SDS-loading buffer and heated at 95 °C for 5 min. Equal amount of proteins were loaded on 10 or 4–20% TGX gels (Bio-Rad), separated by SDS–PAGE and transferred to nitrocellulose membranes (GE Healthcare). Membranes were blocked with 5% fat-free dry milk and incubated overnight at 4 °C with rabbit anti-Diaph3 C-terminal (1:5,000)3 (link), mouse anti-BubR1 (BD Bioscience; catalogue number 612502; Clone 9, 1:500), mouse anti-alpha-tubulin (Sigma; catalogue number T 6199; Clone DM1A; 1:2,000) or chicken anti-GAPDH (Millipore; AB2302; 1:2,000). Relative BubR1 protein quantification was performed using the ImageJ Gel analyser tool. Values were normalized to GAPDH or α-tubulin. The amount of BubR1 protein in controls was set to one. Images of western blottings have been cropped for presentation. Full-size images are presented in Supplementary Fig. 11.

Cardiomyocytes were prepared and treated as above. Cells were lysed with RIPA buffer and 20 μg of protein was loaded onto 10% gels. Membranes were probed for succinate dehydrogenase (ab178423; Abcam), pyruvate dehydrogenase (ab131263; Abcam), aconitase (ab129069; Abcam), and GAPDH (AB2302, 1:5000; Millipore). Densitometric readings of band intensities were obtained using ImageJ software.

HCAEC were seeded at 200 000 cells/well in fibronectin coated 6‐well plates. Cells were pre‐treated for 30 min with the highest working concentrations of each serum profile (19, 20, 44 μM, reflecting 1, 6, and 24 h serum concentrations respectively) or 0.01% DMSO (vehicle control) prior to the addition of 10  ng/mL TNF‐α, and incubated for 15 min at 37°C, 5% CO₂, in a humidified atmosphere. Cells were washed 3× with PBS and lysed with NP‐40 lysis buffer; total protein concentrations were determined by BCA assay, and proteins were separated and probed by SDS‐PAGE and Western blotting, respectively, as described previously by this group 20. Primary antibody solution contained 0.1% PBS with 20% T20 Blocking Buffer (Thermoscientific, UK), with rabbit polyclonal anti‐NFκB p65 (Ser536) antibody (ab28856; Abcam, UK; 1:2000 dilution) and chicken polyclonal anti‐GAPDH (AB2302; Millipore, UK; 1:15 000 dilution) and secondary antibody solutions contained 0.1% PBS with 20% T20 Blocking Buffer and 0.1% SDS, with goat anti‐rabbit (IRdye 800CW; Li‐Cor, UK; 1:15 000 dilution) and donkey‐anti‐chicken (IRdye 680LT; Li‐Cor, UK; 1:15 000 dilution). Membranes were imaged and quantified by densitometry at 700 nm and 800 nm using Odyssey Infrared Imaging System and Odyssey Infrared Imaging System Application Software, respectively (Li‐Cor; version 3.0.21).