Alphaview sa software

Dissociation constant K_d (DNA) was measured using a gel mobility-shift assay that employs a 5′ ³³P-labeled hairpin template (5′-CTCCAGACACGACGCAGTTGCCCGATGGTCGACGTTCGCGAAAGCGAACGTCGACCATCGGGCAACT-3′) blocked at the 3′ end with dideoxy TMP. The radiolabeled hairpin (0.25 nM) was incubated with varying concentrations of wild-type or mutant Taq DNA polymerases (0.5–1000 nM; bracketing the predicted K_d) in 1× Taq buffer at 37°C for 30 min. DNA-protein complexes were run on 10% non-denaturing TBE gels (Life Technologies) in 0.5× TBE buffer. Gels were dried down and exposed to film. The fraction of DNA bound was quantified by densitometry using AlphaView SA software (Alpha Innotech), and then plotted against enzyme concentration to determine K_d values by interpolation.

The cells were lysed with RIPA buffer (Beyotime), and total protein was separated with 10% SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membrances (Millipore, Bedford, MA, USA). The membranes were blocked with tris-buffered saline with tween 20 (TBST) containing 5% skim milk powder and incubated with primary antibodies at 4 °C overnight. After secondary antibody binding, an chemiluminescence reagents kit (New cell & Molecular Biotech, Suzhou, China) was used to detect protein bands. β-actin was used as a control, and the intensity of protein bands was analyzed in AlphaView SA software (Alpha Innotech, CA, USA).

To investigate the expression of putative targets of mmu-miR-152, total protein fractions were purified from allografts and syngrafts as well as normal C3H mice liver. Anti-CaMK II (Cell signaling, USA) and anti-beta-actin (Dawen Biotec, Hangzhou, China) antibody were used for western blot analysis. Equal amounts of protein (40 ug/lane) were resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. After blocking of non-specific binding sites, membranes were incubated overnight at 4°C with Anti-CaMK II antibody (1∶2000), and anti-beta-actin antibody (1∶4000) followed by the corresponding horseradish peroxidase-conjugated secondary antibodies (1∶5000; Dawen Biotec).Then the membranes were developed in the ECL Western detection reagents (Amersham–Pharmacia Biotech, Piscataway, NJ, USA), according to the manufacturer's protocol. The gray value of each band was analyzed and auto-calculated by AlphaView SA software (Alpha Innotech, USA).