polyclonal antibodies against glun2a subunit  (Alomone Labs)

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: A, representative glutamate responses before (Ctrl, black) and during 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 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 baselines 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 (H) at holding potentials of –20 or –70 mV. I and J, pooled data for effects of bath application of 1 μm ALX5407 on TCB‐2‐induced enhancement of NMDAR current amplitude (I) and half‐duration (J) at holding potentials of –70 mV. * P < 0.05, ** P < 0.01 vs. before TCB‐2 application (one‐way repeated‐measures ANOVA followed by a Bonferroni post hoc multiple comparison test). K, representative NMDAR currents in masseter motoneurons before drug application (black), during application of 10 μm TCN 201 (purple) and during addition of 10 μm TCB‐2 to TCN 201 (red) in the presence of 10 μm CNQX, 10 μm glycine and 1 μm TTX, and in the absence of extracellular Mg2+. The baselines of traces before and during drug application were adjusted to be the same. L, pooled data for effects of TCN 201 and TCB‐2 on the amplitude of NMDAR currents. ** P < 0.01 (one‐way repeated‐measures ANOVA followed by a Bonferroni post hoc multiple comparison test). M, representative NMDAR currents in masseter motoneurons before application of ifenprodil and TCB‐2 (black), during application of 10 μm ifenprodil (green) and during addition of 10 μm TCB‐2 to ifenprodil (red) in the presence of 10 μm CNQX, 10 μm glycine and 1 μm TTX, and absence of extracellular Mg2+. The baselines of traces before and during drug application were adjusted to be the same. N, pooled data for effects of ifenprodil and TCB‐2 on the amplitude of NMDAR currents. * P < 0.05 (one‐way repeated‐measures ANOVA followed by a Bonferroni post hoc multiple comparison test). Data are expressed as mean ± SEM.

Article Snippet: After blocking non‐specific binding by incubation with 1% skimmed 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: Membrane, Isolation, Comparison

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: A, representative NMDAR currents in masseter motoneurons during application of 500 μm MCPG (black), and during addition of 10 μm TCB‐2 to MCPG (red) in the presence of 10 μm CNQX, 10 μm glycine and 1 μm TTX, and absence of extracellular Mg2+. The baselines 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 application of 50 μm dl‐TBOA (green), and during 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 baselines 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 repeated‐measures ANOVA followed by a Bonferroni post hoc multiple comparison test).

Article Snippet: After blocking non‐specific binding by incubation with 1% skimmed 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: Comparison