Primary antibodies specific for nr2b

The proteins were isolated from the cells using a radioimmunoprecipitation assay (RIPA) buffer along with a protease inhibitor cocktail, 2 mM phenylmethylsulphonyl fluoride (PMSF), and 1 mM sodium orthovanadate. The cell lysates were centrifuged at 16,000× g for 20 min at 4 °C and the protein was quantified. Equal amounts of proteins were resolved by SDS-PAGE using 6–15% resolving gels and electro-transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% normal horse serum (NHS) in TBS-T and then incubated overnight at 4 °C with primary antibodies specific for NR2B (1:200; Santa Cruz Biotechnology), p-CREB (1:500; Santa Cruz Biotechnology), CREB (1:500; Santa Cruz Biotechnology), cleaved caspase-3 (1:150; Merck Millipore, CA, USA), p53 (1:500; Santa Cruz Biotechnology), and GAPDH (1:1000; Santa Cruz Biotechnology) as well as horseradish peroxidase-conjugated anti-rabbit, anti-mouse, and anti-goat (Santa Cruz Biotechnology). The protein bands were visualized by enhanced chemiluminescence detection (GE Healthcare, Buckinghamshire, UK) and quantified using ImageJ software 1.53t, Bethesda, MD, USA). All the bands were normalized with GAPDH and p-CREB was normalized with CREB.

The proteins were isolated from the cells using a radioimmunoprecipitation assay (RIPA) buffer along with a protease inhibitor cocktail, 2 mM phenylmethylsulphonyl fluoride (PMSF), and 1 mM sodium orthovanadate. The cell lysates were centrifuged at 16,000× g for 20 min at 4 °C and the protein was quantified. Equal amounts of proteins were resolved by SDS-PAGE using 6–15% resolving gels and electro-transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% normal horse serum (NHS) in TBS-T and then incubated overnight at 4 °C with primary antibodies specific for NR2B (1:200; Santa Cruz Biotechnology), p-CREB (1:500; Santa Cruz Biotechnology), CREB (1:500; Santa Cruz Biotechnology), cleaved caspase-3 (1:150; Merck Millipore, CA, USA), p53 (1:500; Santa Cruz Biotechnology), and GAPDH (1:1000; Santa Cruz Biotechnology) as well as horseradish peroxidase-conjugated anti-rabbit, anti-mouse, and anti-goat (Santa Cruz Biotechnology). The protein bands were visualized by enhanced chemiluminescence detection (GE Healthcare, Buckinghamshire, UK) and quantified using ImageJ software 1.53t, Bethesda, MD, USA). All the bands were normalized with GAPDH and p-CREB was normalized with CREB.

SH-SY5Y cells were cultured on poly-D-lysine-coated coverslips (Thermo Fisher Scientific, Waltham, MA, USA) and differentiated into neuronal cells. Following the treatment, the cells were fixed with 4% paraformaldehyde and incubated with primary antibodies specific for NR2B (1:200; Santa-Cruz Biotechnology, CA, USA) and p-CREB (1:200; Santa Cruz Biotechnology) for 90 min at room temperature (RT) followed by incubation with Alexa 555-conjugated donkey anti-mouse IgG (1:500; Molecular Probes Inc., Eugene, OR, USA) and Alexa 488-conjugated donkey anti-rabbit IgG secondary antibodies (1:500; Molecular Probes Inc.), respectively, along with Hoechst 33342 (1:1000; Molecular Probes Inc.) in phosphate-buffered saline (PBS) for 90 min at RT. The cells were then mounted (ProLong Gold anti-fade reagent; Molecular Probes Inc.) and visualized under a Nikon Eclipse Ti2 fluorescence microscope (Nikon, Tokyo, Japan). The images were taken by a DS-Ri2 digital camera (Nikon).