Alphaease image analysis software

Cells were lysed in a lysis buffer [10 mM Tris–HCl (pH 7.4), 1 mM EDTA, and 1% Triton X-100]. Cleared cell lysates were obtained after centrifugation at 10,000× g for 30 min at 4 °C. After measurement of protein concentration using a BCA Protein Assay kit, cell lysates (30–50 μg/lane) were subjected to SDS-PAGE, and separated proteins were electrotransferred to nitrocellulose membranes. The membranes were washed in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 3% bovine serum albumin (BSA). The membranes were incubated overnight at 4 °C in TBS containing 3% BSA and one of the following primary antibodies: p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38 MAPK, p38 MAPK, and β-actin. Subsequently, the labeled proteins were incubated with an HRP-conjugated goat anti-rabbit IgG for 2 h. Blots were developed with the ECL chemiluminescence system and were captured on autoradiographic films (Kodak Image Station 440, Kodak GmbH, Stuttgart, Germany). Films were scanned and densitometric analysis of the bands was performed with AlphaEase Image Analysis Software (Version 5.0.1, Alpha Innotech, San Leandro, CA, USA).

Mice were killed, and different brain tissues, including the hippocampus and the brainstem, were collected. The samples were homogenized in lysis buffer, and the homogenates were centrifuged at 4 °C. The supernatants were collected, and the protein concentrations were determined. The protein samples (20–40 μg) were separated by 10% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subsequently transferred to nitrocellulose membranes. The membranes were blocked with 5% milk in phosphate-buffered saline/0.1% Tween 20 (PBST) for 1 h; then, the membranes were incubated with primary antibodies with shaking at 4 °C overnight. The membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature on the second day. After washing, the protein signals were developed with an ECL chemiluminescence system, and densitometric analysis of the bands was carried out with Alpha Ease Image Analysis Software (version 3.1.2, Alpha Innotech).