rabbit anti nmda receptor subunit 2b  (Cell Signaling Technology Inc)

Journal: Frontiers in Cellular Neuroscience

Article Title: Src Family Kinases Inhibition Ameliorates Hypoxic-Ischemic Brain Injury in Immature Rats

doi: 10.3389/fncel.2021.746130

Figure Lengend Snippet: Activated NR2B (Tyr1472) expression, the enhanced interaction of NR2B with Src, and changes in the levels of p-Src and p-NR2B after the administration of 4-amino-5-(4-chlorophenyl)-7-( t -butyl) pyrazolo [3,4- d ] pyrimidine (PP2) to the immature rat HI brain injury model. (A) Immunoblots showing the levels of multiple proteins. Primary antibodies are listed on the left. (B) Quantification of NR2B (Tyr1472) (p-NR2B) and NR2B levels at 2 h post injury (HI 2 h). N = 7 animals per group. (C) Coimmunoprecipitation was conducted using right hemisphere homogenates with the Src antibody or rabbit IgG as a control. Immune complexes were detected using immunoblotting (IB) with anti-Src and anti-NR2B antibodies. (D) Immunoblots showing the levels of multiple proteins. Primary antibodies are listed on the left. (E) Quantification of the levels of p-Src and Src at HI 2 h. N = 4–6 animals per group. (F) Quantification of the levels of p-NR2B and NR2B at HI 2 h. N = 4–6 animals per group. * p < 0.05 and ** p < 0.01.

Article Snippet: The blots were then blocked with 5% bovine serum albumin in TBS buffer for 1 h at room temperature and probed overnight at 4°C by incubation with the following primary antibodies: Src (1:1,000, CST, catalog number: #2123), phosphor-Src (Tyr 416, 1:1,000, CST, catalog number: #6943), NMDAR subunit NR2B (1:1,000, CST, catalog number: #14544), phospho-NR2B (Tyr 1472, 1:1,000, CST, catalog number: #4208), myelin basic protein (MBP) (1:1,000, Biolegend, SMI99), Iba-1 (1:1,000, Abcam, ab178847), glial fibrillary acidic protein (GFAP) (1:1,000, CST, 12389), β-actin (1:5,000, Absin, abs137975), and GAPDH (1:1,000, Absin, abs132004), followed by appropriate secondary HRP-conjugated antibodies (anti-rabbit, 1:5,000, Absin, abs20002; anti-mouse, 1:5,000, Absin, abs20001).

Techniques: Expressing, Western Blot, Control

Journal: The Journal of Physiology

Article Title: 5‐HT 2A receptor activation enhances NMDA receptor‐mediated glutamate responses through Src kinase in the dendrites of rat jaw‐closing motoneurons

doi: 10.1113/jp275440

Figure Lengend Snippet: Figure 5. Activation of 5-HT2ARs enhances the function of GluN2A-containing NMDARs in masseter motoneurons. A, Representative glutamate responses before (Ctrl, black) and during the application of 10 µM 5-HT (red) in the presence of 1 µM TTX and the absence of extracellular Mg2+. The baselines of traces before and during the 5-HT application were adjusted to be the same. B, Expanded traces of “a”, “b”, and “c” in A. The baselines of traces before and during drug application were adjusted to be the same. C and D, Pooled data for the effects of 5-HT on the glutamate-response amplitude (C) and resting membrane potential (D) in the absence of extracellular Mg2+. * p < 0.05, ** p < 0.01 (paired Student’s t-test). E, Reconstructed morphology of the biocytin-filled masseter motoneuron overlaid with the uncaging spot (white circle). F, In the presence of 10 µM CNQX, 10 µM glycine and 1 µM TTX, and absence of extracellular Mg2+ (black), 10 µM TCB-2 (red) enhanced pharmacologically-isolated NMDAR currents evoked by laser uncaging of MNI-glutamate at holding potentials of –20 mV (top) or –70 mV (bottom). Addition of 20 µM APV to CNQX (green) abolished the NMDAR currents at both holding potentials. The baseline of traces before and during TCB-2 or APV application were adjusted to be the same. G and H, Pooled data for effects of 10 µM TCB-2 on the amplitude of NMDAR currents (G) and half-duration

Article Snippet: After blocking nonspecific binding by incubation with 1% skim milk in a mixture of TBS (pH 7.4) and 0.1% Tween 20 (TBS-T) for 1 h at room temperature, the membranes were incubated in a rabbit monoclonal primary antibody against Src (1:1000; # 2109 Cell Signaling Technology, Beverly, MA, USA), a rabbit monoclonal primary antibody against phospho-Src (p-Src) (Tyr416; 1:1000; # 6943 Cell Signaling Technology), a rabbit polyclonal primary antibody against NMDAR subunit 2A (GluN2A) (1:500; # 07-632 Millipore, Merck KGaA), a rabbit polyclonal primary antibody against phospho-GluN2A (p-GluN2A) (Tyr1325; 1:1000; # ab16646 Abcam, Cambridge, UK), a rabbit polyclonal primary antibody against NMDAR subunit 2B (GluN2B) (1:1000; # ab65783 Abcam), a rabbit polyclonal primary antibody against phospho-GluN2B (p-GluN2B) (Tyr1472; 1:1000; # 4208 Cell Signaling Technology), or a mouse polyclonal anti-β-actin antibody (1:1000; # sc-69879 Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature.

Techniques: Activation Assay, Membrane, Isolation

Journal: The Journal of Physiology

Article Title: 5‐HT 2A receptor activation enhances NMDA receptor‐mediated glutamate responses through Src kinase in the dendrites of rat jaw‐closing motoneurons

doi: 10.1113/jp275440

Figure Lengend Snippet: Figure 7. Metabotropic glutamate receptors or glutamate transporters are not involved in the 5-HT-induced enhancement of the NMDAR current amplitude in masseter motoneurons. A, Representative NMDAR currents in masseter motoneurons during the application of 500 µM MCPG (black), and during the addition of 10 µM TCB-2 to MCPG (red) in the presence of 10 µM CNQX, 10 µM glycine and 1 µM TTX, and the absence of extracellular Mg2+. The baseline of traces before and during drug application were adjusted to be the same. B, Representative NMDAR currents in masseter motoneurons before DL-TBOA and TCB-2 application (black), during the application of 50 µM DL-TBOA (green), and during the addition of 10 µM TCB-2 to DL-TBOA (red) in the presence of 10 µM CNQX, 10 µM glycine and 1 µM TTX, and absence of extracellular Mg2+. The baseline of traces before and during drug application were adjusted to be the same. C and D, Pooled data for effects of MCPG and DL-TBOA on TCB-2-induced enhancement of NMDAR current amplitude (C) and half-duration (D). * p < 0.05, ** p < 0.01, vs. before TCB-2 application (one-way

Article Snippet: After blocking nonspecific binding by incubation with 1% skim milk in a mixture of TBS (pH 7.4) and 0.1% Tween 20 (TBS-T) for 1 h at room temperature, the membranes were incubated in a rabbit monoclonal primary antibody against Src (1:1000; # 2109 Cell Signaling Technology, Beverly, MA, USA), a rabbit monoclonal primary antibody against phospho-Src (p-Src) (Tyr416; 1:1000; # 6943 Cell Signaling Technology), a rabbit polyclonal primary antibody against NMDAR subunit 2A (GluN2A) (1:500; # 07-632 Millipore, Merck KGaA), a rabbit polyclonal primary antibody against phospho-GluN2A (p-GluN2A) (Tyr1325; 1:1000; # ab16646 Abcam, Cambridge, UK), a rabbit polyclonal primary antibody against NMDAR subunit 2B (GluN2B) (1:1000; # ab65783 Abcam), a rabbit polyclonal primary antibody against phospho-GluN2B (p-GluN2B) (Tyr1472; 1:1000; # 4208 Cell Signaling Technology), or a mouse polyclonal anti-β-actin antibody (1:1000; # sc-69879 Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature.

Techniques:

Journal: The Journal of Physiology

Article Title: 5‐HT 2A receptor activation enhances NMDA receptor‐mediated glutamate responses through Src kinase in the dendrites of rat jaw‐closing motoneurons

doi: 10.1113/jp275440

Figure Lengend Snippet: Figure 11. Model of 5-HT2AR-induced enhancement of GluN2A-containing NMDAR function via Src

Article Snippet: After blocking nonspecific binding by incubation with 1% skim milk in a mixture of TBS (pH 7.4) and 0.1% Tween 20 (TBS-T) for 1 h at room temperature, the membranes were incubated in a rabbit monoclonal primary antibody against Src (1:1000; # 2109 Cell Signaling Technology, Beverly, MA, USA), a rabbit monoclonal primary antibody against phospho-Src (p-Src) (Tyr416; 1:1000; # 6943 Cell Signaling Technology), a rabbit polyclonal primary antibody against NMDAR subunit 2A (GluN2A) (1:500; # 07-632 Millipore, Merck KGaA), a rabbit polyclonal primary antibody against phospho-GluN2A (p-GluN2A) (Tyr1325; 1:1000; # ab16646 Abcam, Cambridge, UK), a rabbit polyclonal primary antibody against NMDAR subunit 2B (GluN2B) (1:1000; # ab65783 Abcam), a rabbit polyclonal primary antibody against phospho-GluN2B (p-GluN2B) (Tyr1472; 1:1000; # 4208 Cell Signaling Technology), or a mouse polyclonal anti-β-actin antibody (1:1000; # sc-69879 Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature.

Techniques: