Eck lit

The immunoblotting analyses were conducted as described previously^{40 (link)}. In brief, each specimen was electrophoresed using 12% SDS-polyacrylamide gels. The proteins on the SDS gels were then electrophoretically transferred to nitrocellulose membranes. Following the blocking, the membranes were incubated at 4 °C overnight under gentle agitation with rabbit anti-Akt antibody and rabbit anti-pAkt (Ser 473) antibody (1:1000; Cell Signaling, Danvers, MA, USA), rabbit anti-ERK1/2 (1:1000; Cell Signaling Technology) and rabbit anti-pERK1/2 (1:1000; Cell Signaling Technology). After being washed, the membranes were incubated with horseradish peroxidase (HRP)-labeled anti-rabbit (1:1000; GE Healthcare, Tokyo, Japan) for 2 h at room temperature. Subsequently, the membrane-bound, HRP-labeled antibodies were detected using an enhanced chemiluminescence detection system (ECK lit, GE Healthcare, Tokyo, Japan) with an image analyzer (LAS-1000; Fuji Photo Film, Tokyo, Japan).

SH-SY5Y cells were cultured at a density of 2 × 10⁵ cells/mL. Cells were harvested at each time point with various stimulations, and immunoblotting analyses were conducted. Briefly, each specimen was electrophoresed using 12% SDS-polyacrylamide gels. The proteins on the SDS gels were then electrophoretically transferred to nitrocellulose membranes. Following blocking, the membranes were incubated at 4°C overnight under gentle agitation with each primary antibody: mouse anti-Arc (1 : 1000; Abcam, Heidelberg, Germany) and mouse anti-actin (1 : 5000; Abcam). After washing, the membranes were incubated with horseradish peroxidase- (HRP-) labeled anti-mouse (1 : 2000; GE Healthcare, Buckinghamshire, UK) for 2 h at room temperature. Subsequently, the membrane-bound, HRP-labeled antibodies were detected using an enhanced chemiluminescence detection system (ECK lit; GE Healthcare) with an image analyzer (LAS-1000; Fuji Photo Film; Minato-ku, Tokyo, Japan).