Hippocampal neurons and brain tissue samples were collected for Western blot analysis. Protein samples were separated by 10% SDS–polyacrylamide gel electrophoresis gels and electrotransferred onto 0.45-μm polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat dry milk in tris-buffered saline with Tween (TBST) at RT for 1 hour and then incubated with rabbit anti-GPR40 (1:500; Santa Cruz, CA), anti-NR2B (1:1000; Abcam, Cambridge, UK), anti-NR2A (1:1000; Abcam), mouse anti-GAPDH (Proteintech), or mouse anti–β-actin (Proteintech) antibodies overnight at 4°C. The blots were washed three times and incubated for 1 hour with horseradish peroxidase (HRP)–conjugated anti-mouse or anti-rabbit secondary antibodies (1:5000; Proteintech) in TBST with 5% milk. The blots were then washed in TBST, and bands were visualized using an enhanced chemiluminescence reagent (Thermo, Marina, CA, USA) and the Fusion FX5 image analysis system (Vilber Lourmat, Marne-la-Vallée, France). Relative protein levels were determined by normalization to the GAPDH or β-actin signal using Quantity One software (Bio-Rad, CA, USA).

For coimmunoprecipitation, protein extracts from mouse hippocampal tissues or cultured hippocampal neurons were mixed with immunoprecipitation lysis buffer and incubated with 2 μl of rabbit IgG (Abcam), 4 μl of GPR40, 4 μl of NR2A, or 4 μl of NR2B antibodies overnight at 4°C followed by incubation with 20 μl of protein A/G agarose beads (Santa Cruz) for 2 h at 4°C. The protein-bead complex was then washed five times and collected by centrifugation. The protein samples were mixed with 2× loading buffer and subjected to Western blot analysis with GPR40, NR2A, or NR2B primary antibodies and HRP conjugate mouse anti-rabbit IgG (Conformation Specific, L27A9, Cell Signaling).

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