Las 4000 digital imaging system

The specimens were cut and homogenized in RIPA Buffer with Protease Inhibitor Cocktail (Nacalai tesque, Kyoto, Japan). The homogenate was then centrifuged at 15,000 rpm for 10 min at 4 ˚C. We separated the supernatants of the homogenates using sodium dodecyl sulfate polyacrylamide gel electrophoresis (12.5% e-PAGEL-HR, ATTO) and transferred the proteins onto an Immobilon-P membrane (Clear Blot Membrane-P plus, ATTO). We blotted the membranes with anti-ERK1/2 (#9102, rabbit, polyclonal, 1:1000, Cell Signaling Technology), anti-pERK1/2 (#4370, rabbit, monoclonal, 1:500, Cell Signaling Technology), or anti-β-actin (G043, mouse, 1:1000, abm) antibodies. We treated the sections with a secondary antibody (EnVision+/HRP, Dual Link Rabbit/Mouse, HRP) and visualized the protein bands with a chemiluminescence detection system (Amersham ECL Prime Western Blotting Detection Reagent). We analyzed the signals in the immunoblots using an LAS-4000 digital imaging system (Fujifilm, Tokyo, Japan). We quantified and divided the ERK and p-ERK band intensities by their corresponding loading controls with Image J software (version 1.52u) and determined the ERK phosphorylation rate by p-ERK/ERK.

Western blot analysis of forebrain was performed as previously described41 (link). Briefly, the proteins in the SDS-PAGE gel were transferred onto an Immobilon-P membrane (Millipore, Bedford, MA). Blots were then immunoreacted with anti-ERK1 (Invitrogen, 13–8600), anti-ERK2 (Cell Signaling, #9108), anti-p-ERK1/2 (Cell Signaling, #9101) and anti-β-actin (Cell Signaling, #4967) antibodies diluted in 5% skim milk, and protein bands were visualized using a chemiluminescence detection system (Super Signal West Dura [Pierce, Rockford, IL, USA] or ECL plus [GE Healthcare, Little Chalfont, UK]). Signals in the immunoblots were analyzed by a LAS4000 digital imaging system (Fujifilm, Tokyo, Japan).

Cells were lysed using Radio-Immunoprecipitation Assay (RIPA) Buffer and protein concentration was quantified using Pierce BCA Protein Assay Kit (Pierce Biotechnology, IL, USA). 30 μg of protein lysate was denatured at 70°C for 10 min before electrophoresis on precast 4–12% bis-Tris gels (Life Technologies). Separated proteins were transferred to Immobilin P membranes (Merck Millipore, MA, USA). The membranes were blocked in Tris-buffered saline with 0.1% Tween-20 (TBST) containing 5% BSA and probed with the antibody of interest. The Western Bright Quantum detection kit (Advansta, CA, USA) was used to visualize the detected proteins by a LAS4000 digital imaging system (Fujifilm, Tokyo, Japan). Protein loading was normalized to GAPDH and expression quantified using MultiGauge software (V 3.0, Fujifilm) and mean expression was calculated from three experiments.