Journal: Science Advances

Article Title: CaMKII holoenzyme mechanisms that govern the LTP versus LTD decision

doi: 10.1126/sciadv.abe2300

Figure Lengend Snippet: Quantifications show means ± SEM. * P < 0.05 and ** P < 0.01. Scale bars, 10 μm. ( A ) Schematic of CaMKII movement in response excitatory LTD stimuli. The LTD-induced pT305/306 blocks CaMKII movement to glutamatergic excitatory synapses and instead enables movement to GABAergic inhibitory synapses. ( B ) cLTD stimulation increased surface GABA A R accumulation in WT, but not T305/6AV, CA1 mini-slices (unpaired two-tailed t test, P = 0.0033 for WT and P = 0.6760 for AV, n = 5 and 6 samples (two slices/sample), as detected by immunoblot after surface biotinylation. ( C ) cLTD stimulation decreased surface GluA1 accumulation in WT, but not T305/6AV, CA1 mini-slices (unpaired two-tailed t test, P = 0.0062 for WT and P = 0.6252 for AV, n = 5 and 6 samples (two slices/sample). ( D ) cLTP decreased surface GABA A R cluster size in nonpermeabilized neurons from WT mouse hippocampal cultures (fixed 5 min after washout), while cLTD increased the cluster size (fixed 20 min after washout) (one-way ANOVA, Tukey’s post hoc test versus control, P = 0.0273 for cLTP and P = 0.0046 for cLTD; n = 12, 12, and 13 neurons), as detected by immunocytochemistry. ( E ) By contrast, in nonpermeabilized neurons from T305/5AV hippocampal cultures, cLTD did not increase surface GABA A R clusters. However, cLTP still decreased surface GABA A R clusters ( P = 0.0147 for cLTP and P = 0.7121 for cLTD; n = 15, 13, and 14 neurons).

Article Snippet: The following antibodies were used: GABA A R α1 (1:1000; Synaptic Systems, 224 211), GluA1 (Millipore, AB1504), GluA1 pS845 (1:1000; PhosphoSolutions, p1160-845), GluA1 pS831 (1:1000, PhosphoSolutions, p1160-831), CaMKII pT286 (1:1000, PhosphoSolutions, p1005-286), CaMKII pT305 (1:800; Assay BioTech, A0005; but see fig. S2 and the note below), CaMKII pT306 (1:800; PhosphoSolutions, p1005-306), DAPK1 (1:1000; Sigma-Aldrich, D1319), DAPK1 pS308 (1:1000, Sigma-Aldrich, D4941), GluA1 pS567 (1:1000, provided by K. Roche) , and CaMKIIα (1:5000; CBα2, made in-house), 2° anti-rabbit (1:600; GE Healthcare, NA934V), anti-mouse secondary (1:10,000, GE Healthcare, NA931V), and Alexa Fluor 647 anti-mouse (1:5000; Molecular Probes, A21236).

Techniques: Two Tailed Test, Western Blot, Control, Immunocytochemistry

Journal: Science Advances

Article Title: CaMKII holoenzyme mechanisms that govern the LTP versus LTD decision

doi: 10.1126/sciadv.abe2300

Figure Lengend Snippet: Quantifications show means ± SEM. ** P < 0.01. ( A ) Phosphorylation of the LTD-related GluA1 S567 site by pT286-CaMKII (10 nM kinase subunits) with Ca 2+ /CaM present [stimulated (Stim)] or absent [autonomous (Auton)]. No differences in pS567 were detected between autonomous versus stimulated targeting by either WT or T305/306AV (two-way ANOVA, Bonferroni post hoc test, P = 0.5249 for WT and P = 0.0730 for T305/306AV, n = 5 reactions), although the mutant showed a trend toward increased pS567 with autonomous activity. ( B ) Phosphorylation of the LTP-related GluA1 S831 site by pT286-CaMKII (10 nM kinase subunits) with Ca 2+ /CaM present (stimulated) or absent (autonomous). Enhanced pS831 under stimulated versus autonomous conditions was seen with CaMKII WT (two-way ANOVA, Bonferroni post hoc test, P = 0.0039, n = 5) but not with the T305/306AV mutant ( P = 0.7519, n = 5 reactions).

Article Snippet: The following antibodies were used: GABA A R α1 (1:1000; Synaptic Systems, 224 211), GluA1 (Millipore, AB1504), GluA1 pS845 (1:1000; PhosphoSolutions, p1160-845), GluA1 pS831 (1:1000, PhosphoSolutions, p1160-831), CaMKII pT286 (1:1000, PhosphoSolutions, p1005-286), CaMKII pT305 (1:800; Assay BioTech, A0005; but see fig. S2 and the note below), CaMKII pT306 (1:800; PhosphoSolutions, p1005-306), DAPK1 (1:1000; Sigma-Aldrich, D1319), DAPK1 pS308 (1:1000, Sigma-Aldrich, D4941), GluA1 pS567 (1:1000, provided by K. Roche) , and CaMKIIα (1:5000; CBα2, made in-house), 2° anti-rabbit (1:600; GE Healthcare, NA934V), anti-mouse secondary (1:10,000, GE Healthcare, NA931V), and Alexa Fluor 647 anti-mouse (1:5000; Molecular Probes, A21236).

Techniques: Phospho-proteomics, Mutagenesis, Activity Assay

Journal: Journal of Neurochemistry

Article Title: Pharmacological target sites for restoration of age‐associated deficits in NMDA receptor‐mediated norepinephrine release in brain

doi: 10.1111/jnc.16280

Figure Lengend Snippet: Characterizing the cellular localization of the NMDA receptors regulating [ 3 H] ‐NE release in young rat cortical brain slices. 1 mM Glu‐stimulated‐[ 3 H]‐NE releases in the cerebral cortex tissue slices from young rats ( n = 3–7) in the presence 1 & 3 μM of the TTX, voltage‐gated Na channel blocker, 10 μM MK‐801, and a combination of MK‐801 and TTX. Data are expressed as mean (±SEM) of net fractional release (stimulated—basal), with each data point representing a duplicate from one animal. Data were analyzed using a mixed‐effect analysis followed by Dunnett's multiple comparison test **** p < 0.0001. NE, norepinephrine; Glu, glutamate; TTX, tetrodotoxin.

Article Snippet: The NMDA receptor NR2A and NR2B subunit polyclonal antibodies were used alongside the NR1 monoclonal antibody (PhosphoSolutions, CO, USA; Cat # 1805‐NR1).

Techniques: Comparison

Journal: Journal of Neurochemistry

Article Title: Pharmacological target sites for restoration of age‐associated deficits in NMDA receptor‐mediated norepinephrine release in brain

doi: 10.1111/jnc.16280

Figure Lengend Snippet: The stimulatory effect of Glutamate Vs. NMDA on [ 3 H] ‐NE release in young rat cortical brain slices. In (a), the concentrations‐response curves of glutamate and NMDA‐stimulated [ 3 H] ‐NE releases in the cerebral cortex tissue slices from young rats ( n = 4). In (b), the 1 mM glutamate and NMDA stimulated NE release in the presence and absence of 1.2 mM magnesium in the cerebral cortex tissue slices from young rats ( n = 3). Data were analyzed using an unpaired t ‐test. * p ≤ 0.05, ** p ≤ 0.01 NE, norepinephrine; Glu, glutamate; NMDA, N‐methyl‐ d ‐aspartate; Mg 2+ , magnesium.

Article Snippet: The NMDA receptor NR2A and NR2B subunit polyclonal antibodies were used alongside the NR1 monoclonal antibody (PhosphoSolutions, CO, USA; Cat # 1805‐NR1).

Techniques:

Journal: Journal of Neurochemistry

Article Title: Pharmacological target sites for restoration of age‐associated deficits in NMDA receptor‐mediated norepinephrine release in brain

doi: 10.1111/jnc.16280

Figure Lengend Snippet: Age‐associated changes in the expression of NMDA receptors freely solubilize subunits in the cortical rat tissue homogenate. A representative Western blot for NMDA receptors subunits in the cerebral cortex in (a); and in (b) quantified results for GluN1, GluN2A, and GluN2B expressions in young and aged rats ( n = 5). Data are expressed as mean (±SEM) normalized to young rats, with each data point representing a duplicate from one animal. Data were analyzed using an unpaired t ‐test. * p ≤ 0.05. NMDA, N‐methyl‐ d ‐aspartate).

Article Snippet: The NMDA receptor NR2A and NR2B subunit polyclonal antibodies were used alongside the NR1 monoclonal antibody (PhosphoSolutions, CO, USA; Cat # 1805‐NR1).

Techniques: Expressing, Western Blot

Journal: Journal of Neurochemistry

Article Title: Pharmacological target sites for restoration of age‐associated deficits in NMDA receptor‐mediated norepinephrine release in brain

doi: 10.1111/jnc.16280

Figure Lengend Snippet: Effect of aging on [ 3 H]‐MK‐801 binding to NMDA receptors in the young (2–3 months old) and aged (18–24 months old) rat cortical tissue membrane. In (a) saturation curve of [ 3 H]‐MK‐801 binding to NMDA receptors in young rats in the presence of 10 μM Glu and Gly ( n = 4). The non‐linear least‐squares fitting of the saturation isotherm yielded K d and B max values of 1.8 nM and 970 fmol/mg of protein, respectively. Both total and non‐specific binding of [ 3 H]‐MK‐801 is shown in the curve, and the specific [ 3 H]‐MK‐801 binding is presented with 95% CI in dotted lines. Inset: Saturation data graphed as Scatchard plots. Whereas in (b), the binding of 10 nM [ 3 H]‐MK‐801 in +/− 10 μM Glu and Gly in young and aged rats ( n = 7), each performed in triplicate and repeated twice. In (c), the % increases after subtracting baseline binding from the binding in the presence of 10 μM Glu and Gly. Baseline Binding values represent [ 3 H]‐MK‐801 binding without exogenous addition of Glu and Gly. Data were analyzed using a Mixed‐effect analysis followed by Tukey's multiple comparison tests. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; whereas the % of increase over the baseline was analyzed using an unpaired t ‐test: ** p ≤ 0.01. NMDA, N‐methyl‐d‐aspartate; Glu, Glutamate; Gly, Glycine; K d , dissociation constant; B max , Maximum Binding; n H , Hill Coefficient).

Article Snippet: The NMDA receptor NR2A and NR2B subunit polyclonal antibodies were used alongside the NR1 monoclonal antibody (PhosphoSolutions, CO, USA; Cat # 1805‐NR1).

Techniques: Binding Assay, Membrane, Comparison

Journal: eNeuro

Article Title: Essential Role of Somatic Kv2 Channels in High-Frequency Firing in Cartwheel Cells of the Dorsal Cochlear Nucleus

doi: 10.1523/ENEURO.0515-20.2021

Figure Lengend Snippet: Antibody information

Article Snippet: Kv2.1 (L80/21) , Synthetic peptide 837–853 aa of rat Kv2.1 , Mouse IgG3 mAb , NeuroMab catalogue 75-315, RRID: AB_2315863 , 1 μg/ml.

Techniques: Concentration Assay, Purification