Scion imaging software scion image beta 4

Cells were harvested, washed with PBS, and lysed with RIPA lysis buffer (ATTO Corp., Tokyo, Japan) for 30 min on ice. Protein concentrations were determined by BCA Protein Assay kits (Pierce, Rockford, IL, USA). Equal amounts of protein (20 μg) were resolved by 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. The PVDF membranes were washed with TBST solution (Tween-20 (0.05%), 10 mM Tris-HCl (pH 7.6), and 150 mM NaCl), blocked with skim milk (5%) for 30 min and incubated with the appropriate primary antibody at 4 °C overnight. Each membrane was then incubated with an HRP-conjugated secondary antibody for 2 h. The bands were detected by using the Bio-Rad Chemi Doc™ XRS + System (Bio-Rad, Hercules, CA, USA). The intensity of bands was quantified using Scion imaging software (Scion Image Beta 4.02, Frederick, MD, USA).

Cells were harvested, washed twice with PBS, and lysed with buffer (20 mM Tris (pH 7.5), 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid, 1% Triton X-100, 1 mg/ml aprotinin, and 1 mM phenylmethylsulfonylfluoride) for 30 min on ice. The lysates were then cleared by centrifugation (22 250 × g at 4 °C for 30 min). Protein concentration was determined by the Bradford method.^{57 (link)} Equal amounts of protein (20 μg) were resolved by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membranes. The membranes were washed with Tris-buffer solution-Tween 20 solution (10 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.05% Tween-20), blocked with 5% skim milk for 1 h, and incubated with appropriate primary antibody at 4 °C for overnight. The membrane was then washed and detected with a horseradish peroxidase-conjugated secondary antibody. The bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech Inc., Buckinghamshire, UK). The relative optical density of the bands was quantified using the Scion Imaging Software (Scion Image Beta 4.02, Frederick, MD, USA).