Ab81271

The primary antibodies used were anti-GAPDH (1:2000 abcam ab8245), anti-NR2A(1:1000 abcam ab124913), anti-NR2B(1:2000 abcam ab81271), anti-NR1(1:2000 abcam ab109182), anti-VGLUT1(1:2000 abcam ab180188). The secondary antibodies used were anti-mouse IgG HRP(1:5000 promega W4208) and anti-rabbit IgG HRP(1:5000 promega W4011). The brands were detected with ECL Western Blotting Substrate (promega w1001).

Protein samples were collected from neurons cultured in 60 mm dishes (4.5 × 10⁶/well) that were treated with lysis buffer (50 mM Tris–HCl pH 7.5, 250 mM NaCl, 10 mM EDTA, 0.5%, NP-40, 10 μM leupeptin, 1 mM PMSF, and 4 mM NaF). Thirty micrograms of total protein was separated by 10% SDS-PAGE and then was transferred to PVDF membranes (Millipore, CA, United States). The membranes were blocked with 5% skim milk and then were incubated with primary antibodies against GluN2A (1:400, ab227233, Abcam, United Kingdom), p-GluN2B (1:200, ab81271, Abcam, United Kingdom), GluN2B (1:400, ab65783, Abcam, United Kingdom), p-DAPK1 (1:200, D4941, Sigma, United States), DAPK1 (1:400, 3008S, Cell Signaling Technology, United States), p-ERK (1:200, 4370S, Cell Signaling Technology, United States), ERK (1:200, sc93, Santa Cruz Biotechnology, United States) and β-tubulin (1:1000, K106281P, Solarbio, China). After washing with TBS-T for 5 min for a total of 3 times, HRP-conjugated secondary antibodies (goat anti-rabbit IgG, rabbit anti-goat IgG, and goat anti-mouse IgG) were applied to the blots. The blots were developed using ECL Western blotting detection reagents (Thermo Fisher Scientific, Waltham, MA, United States). The band densitometry was analyzed using an Amersham Imager 600 system.

The tissue samples were lysed with RIPA lysate containing protease inhibitor and phosphatase inhibitor (Beyotime). The protein concentration was determined using a DC protein analysis kit (500-0111; Bio-Rad, Hercules, CA, USA) at 750 nm. The proteins were separated by sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membrane. The membrane was then blocked with 5% bovine serum albumin for 1 h and probed with rabbit anti-human antibodies (1 : 1000) overnight at 4°C. Next, the membrane was reprobed with secondary antibody (1 : 5000). Luminata Western horseradish peroxidase (HRP) substrate (Millipore, Billerica, MA, USA) and Kodak XBT-1 negatives were adopted for color development. The band was quantified using Bio-Rad Quantity One software, normalized to actin level. All results were normalized with blank control values of 100%.
The antibodies used were as follows: GABA_B receptor (Invitrogen, #PA1-32248), NR2B (Abcam, Cambridge, UK; ab81271), PKC (Abcam, ab31), CaMKII (Abcam, ab171095), phosphorylation (p)-mitogen-activated protein kinase (ERK1/2) (Abcam, ab54230), p-CREB (Abcam, ab32096), and beta-actin antibody (Abcam, ab8226). Secondary antibody was mouse anti-rabbit immunoglobulin G (IgG) light chain (Abcam, ab8226).