mouse anti-glua2 n-terminal  (NeuroMab)

Journal: International Journal of Molecular Sciences

Article Title: Daily Brief Heat Therapy Reduces Seizures in A350V IQSEC2 Mice and Is Associated with Correction of AMPA Receptor-Mediated Synaptic Dysfunction

doi: 10.3390/ijms24043924

Figure Lengend Snippet: Surface GluA2 AMPA receptor in neonatal hippocampal neurons is decreased in A350V neurons and is increased after heat treatment. ( A ) Representative images (20× magnification) of total (T.GluA2) and surface (S.GluA2) GluA2 AMPA receptor in wild-type (WT) and mutant (A350V) IQSEC2 hippocampal neurons. Neurons treated with heat (40 °C) for 1 h are labelled as MUT.H.S or WT.H.S. ( B ) Quantification of relative surface GluA2 expression (ratio: S.GluA2/T.GluA2). Data are reported as the mean ± SEM obtained from at least 4 neuronal culture preparations for each of the 4 groups. ** p < 0.01, *** p < 0.001, n.s. p > 0.05.

Article Snippet: To label neuronal surface GluA2 AMPAR, coverslips were incubated at 4 °C overnight in PBS containing 3% normal goat serum and mouse monoclonal extracellular anti-N-terminal GluA2 IgG (Synaptic System, Gottingen, Germany, cat# 182111).

Techniques: Mutagenesis, Expressing

Journal: Molecular Psychiatry

Article Title: IQSEC2 mutation associated with epilepsy, intellectual disability, and autism results in hyperexcitability of patient-derived neurons and deficient synaptic transmission

doi: 10.1038/s41380-021-01281-0

Figure Lengend Snippet: a A representative trace of excitatory postsynaptic currents (EPSCs) that were measured in a control neuron at 5 weeks post-differentiation. b A representative trace of EPSCs measured in an IQSEC2 -mutant neuron at 5 weeks post-differentiation. c The average amplitude of synaptic events was increased in the IQSEC2 -mutant neurons ( p = 0.053). d The average rate of synaptic events was not significantly different between control and IQSEC2 -mutant neurons. e The cumulative distribution of the amplitude of synaptic events is right-shifted in the IQSEC2 -mutant neurons indicating higher amplitudes of synaptic events. f A representative trace of synaptic activity measured in a control neuron at 7 weeks of age. g A representative trace of synaptic activity measured in an IQSEC2 -mutant neuron at 7 weeks. h The average amplitude of synaptic events is similar between IQSEC2 -mutant and control neurons at 7 weeks of age. i The average rate of synaptic events was not significantly different between control and IQSEC2 -mutant neurons at 7 weeks. j The cumulative distribution of the amplitude of synaptic events shows a slight right shift in IQSEC2 -mutant neurons. k A representative trace of synaptic activity measured in a control neuron at 11 weeks. l A representative trace of synaptic activity measured in an IQSEC2 -mutant neuron at 11 weeks. m The average amplitude of synaptic events is similar between IQSEC2 -mutant and control neurons at 11 weeks post-differentiation. n The average rate of synaptic events is significantly decreased in IQSEC2 -mutant neurons compared to control neurons at 11 weeks. o The cumulative distribution of the amplitude of synaptic events. p Immunoblots after BS 3 -crosslinking of samples to assess surface (crosslinking “+”) and total protein expression of AMPARs (crosslinking “−”). q Quantification of relative total protein levels of AMPA receptor subunits in samples not cross-linked. r Quantification of relative surface protein levels of AMPA receptor subunits in samples treated with BS 3 (crosslinking “+”). Surface expression is the ratio of s- GluA2 to the total GluA2 signal in the lane. Signal intensities normalized to actin signal and wild type as 100%. s Representative images of labeled total (t GluA2 ) and surface (s GluA2 ) GluA2 in IQSEC2 -mutant and control DG granule neurons. t Quantification of the percentage of s GluA2 to t GluA2 as a measure surface GluA2 expression. u (left). An image of the FACS gating of a control neuronal culture at 7 weeks after the start of differentiation. The live neurons that passed all the gating (GFP positive indicating PROX1 expression and live) were ~55% of the cells in the culture (marked with the red line). u (right). An image of the FACS gating of an IQSEC2 -mutant neuronal culture at 7 weeks. The live neurons that passed all the gating were ~41% of the cells in the culture. v (left). An image of the FACS gating of a control neuronal culture at 11 weeks. The live neurons that passed all the gating were approximately 36% of the cells in the culture. v (right) An image of the FACS gating of an IQSEC2 -mutant neuronal culture at 11 weeks. The live neurons that passed all the gating were approximately 14% of the cells in the culture. w (left) Immunohistochemistry staining for DAPI (blue), MAP2 (green), and Caspase3 (red) in an example control neuronal culture. w (right) Immunohistochemistry staining for DAPI (blue), MAP2 (green), and Caspase3 (red) in an example IQSEC2 -mutant neuronal culture. x An increased average percentage of neurons stained for caspase3 (out of the total MAP2 positive neurons) in the IQSEC2 -mutant neuronal cultures compared to the control neuronal cultures indicate an increased number of apoptotic neurons. In this figure, asterisks indicate statistical significance by the following code: * p value < 0.05, ** p < 0.01. Error bars represent the standard error.

Article Snippet: To label surface GluA2 , sections were incubated at 4 °C overnight in PBS containing 0.5% normal goat serum and a mouse monoclonal Alexa Fluor 488-conjugated extracellular anti-N-terminal GluA2 antibody (Millipore-Sigma, MAB397A4).

Techniques: Control, Mutagenesis, Activity Assay, Western Blot, Expressing, Labeling, Immunohistochemistry, Staining