Neuronal cell culture and Western blotting

Primary hippocampal cultures were established from postnatal day 0 mice as described (Zhou et al. 2009). The mGluR1/5-mediated activation of ERK1/2, S6K1, and Arc expression was determined with neurons at DIV 8 (days in vitro 8). Control and treated neurons were lysed and proteins were extracted in Laemmli buffer. Same amount of cell extract was separated by SDS-PAGE followed by transferring to nitrocellulose membranes. The following antibodies were used to detect the corresponding targets. Arc antibody was obtained from Synaptic Systems (Cat # 156003, 1:1000 dilution). β-actin antibody was from Sigma (Cat #A5441, 1:10,000 dilution). Anti-phospho-ERK1/2 (Cat #9101, 1:1000 dilution), anti-ERK1/2 (Cat #9102, 1:1000 dilution), anti-S6K1 (Cat #9202, 1:1000 dilution), and anti-phospho-S6K1 (at Thr421/Ser424) (Cat #9204, 1:1000 dilution) were from Cell Signaling.

For quantification purpose, the level of Arc was normalized to the level of β-actin. As the phosphorylation of both ERK1 and ERK2 was responsive to mGluR1/5 agonist DHPG and various inhibitors, the level of pERK1 and pERK2 was combined, quantified, and normalized to total ERK1 and ERK2. The level of phosphorylated S6K1 was normalized to the level of total S6K1. The relative intensity of the Western blot signal in the no treatment control group, which was quantified using ImageJ (NIH, MD, USA), was defined as 1. The signal in the treatment samples was normalized to the control group.