Protein from BMDMs lysates was resolved NuPAGE 4%-12% gels (Thermo Fisher Scientific) or Tris–Glycine gel, and transferred to PVDF membranes (Millipore, Burlington, MA, USA). After blocking with 5% skim milk, the membranes were incubated with primary antibodies at 4 °C overnight. Primary antibodies against the following proteins: iNOS, HIF-1α (Novus, Centennial, CO, USA), NMDAR1 (Abcam), p-p65 (S536), p65, p-PI3K (Y458), PI3K, p-AKT (T308), AKT, p-p70S6K (T389), p70S6K, and LDHA (Cell Signaling Technology, Danvers, MA, USA), and Actin (Sigma). The HRP-conjugated secondary antibodies were used to incubate the membranes for 2 h at room temperature. Membranes were detected using Image Quant LAS-4000 (GE Healthcare, Chicago, IL, USA).
Nmdar1
Manufactured by Abcam
Sourced in United States
NMDAR1 is a subunit of the N-methyl-D-aspartate receptor (NMDAR), a type of ionotropic glutamate receptor that is essential for excitatory neurotransmission in the central nervous system. The NMDAR1 subunit is a key component of the NMDAR complex and plays a critical role in receptor function and signaling.
Automatically generated - may contain errors
Lab products found in correlation
Primary antibody dilution buffer, by Beyotime (2 mentions)
Nmdar2a, by Abcam (2 mentions)
Anti ferritin, by Abcam (2 mentions)
Rasd1, by Abcam (2 mentions)
Superoxide dismutase 2 sod2, by Abcam (2 mentions)
Mitoferrin1, by Abcam (2 mentions)
Anti irp2, by Alpha Diagnostic (2 mentions)
Anti dmt1, by Alpha Diagnostic (2 mentions)
Peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Anti tfr1, by Zymo Research (2 mentions)
Leica tcs sp2 laser scanning confocal microscope, by Leica Microsystems (2 mentions)
P p70s6k, by Cell Signaling Technology (1 mentions)
Nupage 4 12 gel, by Thermo Fisher Scientific (1 mentions)
Hif 1α, by Novus Biologicals (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
P70s6k, by Cell Signaling Technology (1 mentions)
P pi3k, by Cell Signaling Technology (1 mentions)
Actin, by Merck Group (1 mentions)
6 protocols using nmdar1
1
Western Blot Analysis of Protein Expressions
2
Quantitative Protein Analysis of Hippocampal Samples
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Proteins were obtained from the hippocampal tissue or cultured neurons or isolated mitochondria. For western blotting, 35–50 μg protein was added per lane of 12% SDS-PAGE. Primary antibodies were diluted in primary antibody dilution buffer (Beyotime Biotech., Shanghai, China). Antibodies used included the following: anti-GAPDH (Abgent Biotech. Co. Ltd., Suzhou, China), anti-ferritin, superoxide dismutase 2 (SOD2), Mitoferrin1, Drp1, Mfn2, RASD1, NMDAR1, and NMDAR2A (Abcam, Cambridge, MA, USA), anti-TfR1 (Zymed, San Francisco, CA, USA), anti-IRP2 (polyclonal, raised from rabbit), and anti-DMT1 (Alpha Diagnostic International, San Antonio, TX, USA). Detection was performed using peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, UK). Quantification of the density of the western bands was done with programme ImageJ (http://rsb.info.nih.gov/ij/ ).
3
Immunofluorescence Analysis of Dopamine-Induced Signaling
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PCAs were seeded and cultured on glass coverslips precoated with 0.01% poly-l-lysine (Sigma-Aldrich) for 1 h. Following treatment of the cells with DA (final concentration of 5 or 50 μM) for 24 h, they were fixed with 4% paraformaldehyde for 30 min and then treated with 0.1% Triton X-100 for 10 min at room temperature.
Blocking was achieved with PBS containing 5% normal goat serum for 1 h at room temperature. Sections were then incubated overnight at 4°C with the following primary antibodies: NMDAR1 (1:100; Abcam), CaM (1:100; Abcam), nNOS (1:50; Abcam), sGC (1:50; Abcam) and cGMP (1:50; Santa Cruz Biotechnology, Inc.). Binding of primary antibodies was detected by incubating the sections for 30 min with Alexa Fluor 594 (red) conjugated secondary antibody. Imaging was performed with a Leica TCS SP2 confocal laser scanning microscope (Leica Microsystems). The image data were analyzed and quantified using ImagePro Plus software (Media Cybernetics, Inc.) (22 (link)).
Blocking was achieved with PBS containing 5% normal goat serum for 1 h at room temperature. Sections were then incubated overnight at 4°C with the following primary antibodies: NMDAR1 (1:100; Abcam), CaM (1:100; Abcam), nNOS (1:50; Abcam), sGC (1:50; Abcam) and cGMP (1:50; Santa Cruz Biotechnology, Inc.). Binding of primary antibodies was detected by incubating the sections for 30 min with Alexa Fluor 594 (red) conjugated secondary antibody. Imaging was performed with a Leica TCS SP2 confocal laser scanning microscope (Leica Microsystems). The image data were analyzed and quantified using ImagePro Plus software (Media Cybernetics, Inc.) (22 (link)).
4
Immunofluorescence Antibody Staining Protocol
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5
Immunohistochemical Analysis of Cerebral Cortex
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Four-micron frozen cerebral cortex sections fixed in acetone or 4% formaldehyde were blocked for endogenous peroxidase activity with 0.03% H2O2 if appropriate. Blocking was achieved with phosphate-buffered saline (PBS) containing 5% normal goat serum (Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 1 h at room temperature. Sections were then incubated overnight at 4°C with the following primary antibodies; NMDAR1 (1:100; mouse monoclonal; Abcam, Cambridge, MA, USA), CaM (1:100; mouse monoclonal; Abcam), nNOS (1:50; Rabbit monoclonal; Abcam), sGC (1:50; rabbit polyclonal; Abcam), cGMP (1:50; mouse monoclonal; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and GFAP (1:50; rabbit polyclonal/mouse monoclonal; Abcam). Binding of primary antibodies was detected by incubating the sections for 30 min with fluorescein isothiocyanate (FITC) (green)/Alexa Fluor 594 (red) conjugated secondary antibody. Imaging was performed with a Leica TCS SP2 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). The image data were analyzed and quantified using ImagePro Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).
6
Protein Analysis in Hippocampus and Neurons
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Proteins were obtained from hippocampal tissue or cultured neurons or isolated mitochondria. For western blotting, 35-50 µg protein was added per lane of 12% SDS-PAGE. Primary antibodies were diluted in primary antibody dilution buffer (Beyotime Biotech., Shanghai, China). Antibodies used included: anti-GAPDH (Abgent Biotech. Co. Ltd., Suzhou, China), anti-ferritin, superoxide dismutase 2 (SOD2), Mitoferrin1, Drp1, Mfn2, RASD1, NMDAR1, and NMDAR2A (Abcam, Cambridge, MA, USA), anti-TfR1 (Zymed, San Francisco, CA, USA), anti-IRP2 (polyclonal, raised from rabbit), and anti-DMT1 (Alpha Diagnostic International, San Antonio, TX, USA). Detection was performed using peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, UK). Quantification of the density of the western bands was done with programme ImageJ (http://rsb.info.nih.gov/ij/).
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