Western Blotting

Collected surface proteins were suspended in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred electrophoretically onto polyvinylidene fluoride membranes (Millipore). After blocking with 2% BSA in TBS buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl), the membranes were incubated with an anti-PAC1 antibody (1:1,000 dilution), anti-5-HT_1A antibody (1:1,000 dilution), anti-5-HT_2A antibody (1:1,000 dilution), anti-D2 antibody (1:1,000 dilution), anti-mGlu2/3 antibody (1:1,000 dilution), anti-β-actin antibody (1:2000 dilution) or anti-alpha 1 sodium potassium ATPase antibody (1:1000 dilution) overnight at 4°C. After incubation with a horseradish peroxidase-conjugated anti-rabbit IgG (1:2,000 dilution) or anti-mouse IgG (1:2,000 dilution) secondary antibody for 1 h at room temperature, proteins were detected by chemiluminescence and visualized with an ImageQuant LAS 4000 system (GE Healthcare, Little Chalfont, UK). For quantification, the bands of specific immune-complexes were analyzed using ImageJ software.