Scion image alpha 4

Total cell extract was prepared as previously described [22 (link)]. After protein content determination, the cell lysate was separated using NuPAGE Bis-Tris precast 10% polyacrylamide gels under reducing conditions (Invitrogen, Carlsbad, CA, USA). Resolved proteins were transferred onto nitrocellulose sheets (Amersham, Freiburg, Germany), and the resulting membranes were saturated with a 5% blocking agent (Amersham) in Tris-buffered saline (TBS, 20 mmol/L Tris (pH 7.6) and 132 mmol/L NaCl) for 1 h at room temperature. Membranes were then incubated overnight at 4°C with primary antibodies against human ATP5β, PGC-1α, NRF-1, TFAM, and β-actin, followed by a horseradish peroxidase-conjugated secondary antibody. The enhanced chemiluminescence (ECL) method (Amersham) was used to reveal positive bands, according to the manufacturer's instructions. Bands were analysed quantitatively using the Scion Image Alpha 4.0.3.2 software (Scion Corporation).

As reported earlier, 300 μg of protein from brain cortex of 17-day-old control, Cdk5 cKO2, and dKO mice, were dissolved in T-PER buffer, and they were immunoprecipitated using 4 μg of anti-Cdk5 (C8) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) [21 (link)]. Immunoprecipitated proteins (IP) were washed three times in cold PBS, and twice in kinase buffer (20 mM Tris HCl (pH 7.4), 10 mM MgCl₂, 1 mM EDTA). Then, the IP was mixed with the kinase assay mixture (100 mM Tris HCl (pH 7.4), 50 mM MgCl₂, 5 mM EDTA, and 5 mM DTT) plus 5 units of (γP³²)-ATP, and using 5 μg of histone H1 as a substrate. The kinase assays were carried out for 30 minutes at 30°C, and the kinase activity reaction was stopped by adding 5× sodium dodecyl sulfate sample buffer and boiling for ten minutes at 70°C. Kinase reaction was electrophoresed on a 4 to 20% polyacrylamide gel, and then gels were exposed to X-ray films, for 1 to 3 hours at -80°C. The incorporation of P³² to histone H1 was quantified to measure band intensity using Scion Image Alpha 4.0.3.2 software (Scion Corporation, Frederick, MD, USA).