The mouse myocardial tissues were harvested to extract the nucleoproteins mentioned above and separated on 8/12% sodium dodecyl sulphate polyacrylamide gels through electrophoresis and blotted onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). These PVDF blots were probed against rabbit polyclonal antibodies [acetylated lysine 9 on histone H3 (H3K9ac), atrial natriuretic peptide (ANP), foetal isoform of myosin heavy chain (β‐MHC), α‐actin, p300 and PCAF] (Abcam, Cambridge, UK, 1:1,000 dilution) or rabbit polyclonal antibody against histone H3 and β‐actin (Beyotime, Shanghai, China, 1:1,000 dilution) in Tris‐buffered saline with Tween 20 (TBST) plus 5% non‐fat milk at 4°C overnight. HRP‐conjugated goat anti‐rabbit antibody (Santa Cruz Biotechnology, Texas, USA) was used as the secondary antibody. After the PVDF membranes were scanned, the bands were subjected to analysis using the Quantity One (Version 4.4) software package (Bio‐Rad, CA, USA).
Quantity one version 4.4
Manufactured by Bio-Rad
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Quantity One (Version 4.4) is a software application developed by Bio-Rad for quantitative analysis of 1D and 2D electrophoresis gels, Western blots, and other types of biological images. It provides tools for image capture, analysis, and data management. The software is compatible with various Bio-Rad and third-party imaging systems.
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5 protocols using quantity one version 4.4
1
Histone Protein Expression Analysis in Mouse Myocardium
2
Quantifying Cardiac Protein Levels
3
Quantitative Analysis of cTnI and acH3 Levels
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Western blotting assays for cTnI and acetylated histone 3 (acH3) were performed as previously described 8 , 14 . An anti‐cTnI monoclonal antibody (cTnI) that recognized mouse cTnI was used at a dilution of 1:1000 (Abcam; Cambridge, UK, ab47003), anti‐acH3 (MILLIPORE 17‐615) and total histone 3 (H3) monoclonal antibodies (ab1791) were used at a dilution of 1:2000, and anti‐β‐actin monoclonal antibody (BOSTER, Wuhan, China) was used at a dilution of 1:1000. The immune‐reactive protein bands were visualized with Chemiluminescence Luminol Reagent (Merck Millipore, USA). After scanning, protein bands were analysed with Quantity One version 4.4 software (Bio‐Rad, CA, USA).
4
Western Blot Protein Detection
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(TBST) containing 5% non-fat milk, and incubations were performed at 4 °C overnight. HR-conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology, Texas, USA, 1:2000 dilution) was used as the secondary antibody. After scanning, the bands were subjected to analysis using the Quantity One (Version 4.4) software package (Bio-Rad, CA, USA).
5
Microsatellite Markers for Peruvian Scallop
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A total of 31 microsatellite markers developed for the bay scallop (referred to Roberts et al., 2005; Zhan et al., 2005 Zhan et al., , 2006) ) were assessed in Peruvian scallop to select the common markers. The chosen markers were then used for SSR analysis in the hybrids and parental groups. The annealing temperature for these analyses ranged from 50° to 65°C. PCR amplification was performed according to the protocol described by Zhan et al. (2005) . PCR products were detected by electrophoresis on 10% non-denaturing polyacrylamide gel in 1X TBE buffer. The gels were stained with ethidium bromide and visualized under ultraviolet light (Zhan et al., 2005) . The genotypes of different loci were scored with the software QUANTITY ONE version 4.4 (Bio-Rad) by comparing with the DNA molecular standard (100-bp DNA ladder marker, TaKaRa).
The level of genetic diversity per group was evaluated by the number of alleles (N) and the effective number of alleles per locus (A E ), as well as the observed (H O ) and expected (H E ) heterozygosities using POPGENE version 1.31 (Yeh et al., 1999) . The polymorphic information content (PIC) value was estimated according to the following formula:
where n is the allele number, and P i and P j are the frequencies of the i th and j th alleles.
The level of genetic diversity per group was evaluated by the number of alleles (N) and the effective number of alleles per locus (A E ), as well as the observed (H O ) and expected (H E ) heterozygosities using POPGENE version 1.31 (Yeh et al., 1999) . The polymorphic information content (PIC) value was estimated according to the following formula:
where n is the allele number, and P i and P j are the frequencies of the i th and j th alleles.
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