Journal: Journal of Neuroscience

Article Title: Regulation of Nucleus Accumbens Activity by the Hypothalamic Neuropeptide Melanin-Concentrating Hormone

doi: 10.1523/jneurosci.5858-09.2010

Figure Lengend Snippet: Figure 2. MCH acts via Gi/o to reduce GluR1 pSer 845 in the AcbSh. Graphs represent the ratio of phosphorylated signal to total protein signal for all treatments. A, Basal or SKF 81297-mediated GluR1 pSer 845 reduction by MCH is blocked by in vivo PTX treatment (n 12–15 slices per group). B, MCH-mediated reduction is not affected by inhibition of PLC–PKC pathway with U73122 (n 5 slices per group). *p 0.05, **p 0.01. Error bars indicate SEM.

Article Snippet: For GluR1 protein detection, membranes were first blotted with the phospho-antibodies at 1:500 (rabbit polyclonal, GluR1 Ser 845 and Ser 831 antibodies from PhosphoSolutions).

Techniques: In Vivo, Inhibition

Journal: Journal of Neuroscience

Article Title: Regulation of Nucleus Accumbens Activity by the Hypothalamic Neuropeptide Melanin-Concentrating Hormone

doi: 10.1523/jneurosci.5858-09.2010

Figure Lengend Snippet: Figure4. SurfacelevelsofGluR1andmEPSCamplitudearereducedbyMCHinMSNsoftheAcbSh.A,Datarepresenttheratioof surface(cross-linked)tointernal(un-cross-linked)GluR1signal,andisnormalizedtountreatedcontrol.Treatmentconditionsare indicated below the graph along with representative surface and internal GluR1 signal (n 7–10 slices per group). B, C, Repre- sentativetracesbefore(B)andafter(C)bathapplicationofMCH(3M).D,E,Cumulativeprobabilityhistogramforamplitude(D) and interevent interval (E). n 8 neurons for mEPSC recordings; *p 0.05., **p 0.01. Error bars indicate SEM.

Article Snippet: For GluR1 protein detection, membranes were first blotted with the phospho-antibodies at 1:500 (rabbit polyclonal, GluR1 Ser 845 and Ser 831 antibodies from PhosphoSolutions).

Techniques:

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: eLife

Article Title: Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model

doi: 10.7554/eLife.89453

Figure Lengend Snippet: ( A, B ) Hippocampal neurons were subjected to ( A ) chemical LTP (cLTP) or ( B ) chemical LTD (cLTD). DKK3 levels were analyzed in the supernatant (extracell) and cell lysates by western blot. Phospho-GluA1 Ser845 was used as a readout of LTP and LTD induction, and tubulin as a loading control for the cell lysate. Graphs show the levels of DKK3 relative to control and the ratio of extracellular/lysate DKK3 levels (Mann-Whitney Test, n=3 independent cultures for cLTP and Student’s T-test, n=6 independent cultures for cLTD). ( C ) Representative immunoblots show DKK3 levels in the cell lysate and extracellular (extracell) fraction of acute WT hippocampal slices treated with vehicle (Ctrl) or NMDA (cLTD) and/or Brefeldin A (BFA) for 60 min. Actin was used as a loading control in homogenates. Graphs show densiometric quantifications of DKK3 relative to the control condition and the ratio of extracellular/lysate DKK3 levels (Kruskal-Wallis followed by Dunn’s multiple comparisons; n=5 animals). ( D ) Immunoblot showing DKK3 is less abundant in the extracellular fraction when compared to the cell lysate fraction. Representatives immunoblot of J20 brain slices treated with control or APV for 3 hr. Time exposure for obtaining DKK3 chemiluminescent images is indicated. Figure 2—figure supplement 3—source data 1. Uncropped western blot gels. Raw and annotated WB images. The representative western blot images for are indicated within a blue square.

Article Snippet: Primary antibodies and dilutions used for western blot were: Mouse Anti- Aβ (6E10 clone, 1:1000, Biolegend, Cat# 803001, RRID: AB_2564653 ), goat anti- Mouse DKK-3 (1:1000, R and D Systems, Cat# AF948, RRID: AB_355734 ), goat anti- Human DKK-3 (1:1000, R&D Systems, Cat# AF1118, RRID: AB_354610 ), rabbit anti-GAPDH (1:5000, Abcam, Cat# ab181602, RRID: AB_2630358 ), rabbit anti-GluA1 (1:1000, Cell Signaling Technology, Cat# 13185, RRID: AB_2732897 ), rabbit anti-phospho GluA1 Serine 845 (1:1000, Cell Signaling Technology, Cat# 8084, RRID: AB_10860773 ), rabbit anti- SAPK/JNK (1:1,000, Cell Signaling Technology, Cat# 9252, RRID: AB_2250373 ), mouse anti-Phospho-SAPK/JNK (Thr183/Tyr185) (1:500, Cell Signaling Technology, Cat# 9255, RRID: AB_2307321 ), mouse anti-Tubulin (1:5000, Sigma-Aldrich, Cat# T9026, RRID: AB_477593 ), mouse Anti-Vinculin (1:2000, Sigma Aldrich, Cat# v4505, RRID: AB_477617 ), HRP mouse anti-beta Actin (1:10,000, Abcam, Cat# ab8224, RRID: AB_449644 ).

Techniques: Western Blot, Control, MANN-WHITNEY

Journal: eLife

Article Title: Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model

doi: 10.7554/eLife.89453

Figure Lengend Snippet:

Article Snippet: Primary antibodies and dilutions used for western blot were: Mouse Anti- Aβ (6E10 clone, 1:1000, Biolegend, Cat# 803001, RRID: AB_2564653 ), goat anti- Mouse DKK-3 (1:1000, R and D Systems, Cat# AF948, RRID: AB_355734 ), goat anti- Human DKK-3 (1:1000, R&D Systems, Cat# AF1118, RRID: AB_354610 ), rabbit anti-GAPDH (1:5000, Abcam, Cat# ab181602, RRID: AB_2630358 ), rabbit anti-GluA1 (1:1000, Cell Signaling Technology, Cat# 13185, RRID: AB_2732897 ), rabbit anti-phospho GluA1 Serine 845 (1:1000, Cell Signaling Technology, Cat# 8084, RRID: AB_10860773 ), rabbit anti- SAPK/JNK (1:1,000, Cell Signaling Technology, Cat# 9252, RRID: AB_2250373 ), mouse anti-Phospho-SAPK/JNK (Thr183/Tyr185) (1:500, Cell Signaling Technology, Cat# 9255, RRID: AB_2307321 ), mouse anti-Tubulin (1:5000, Sigma-Aldrich, Cat# T9026, RRID: AB_477593 ), mouse Anti-Vinculin (1:2000, Sigma Aldrich, Cat# v4505, RRID: AB_477617 ), HRP mouse anti-beta Actin (1:10,000, Abcam, Cat# ab8224, RRID: AB_449644 ).

Techniques: Isolation, Knock-Out, Control, Sequencing, shRNA, Recombinant, TUNEL Assay, cDNA Synthesis, Fluorsave, Software, Microscopy, Immunofluorescence, Patch Clamp

Journal: Journal of neurochemistry

Article Title: Dopamine and cyclic AMP-regulated phosphoprotein-32 phosphorylation pattern in cocaine and morphine-sensitized rats.

doi: 10.1111/j.1471-4159.2004.02510.x

Figure Lengend Snippet: Fig. 2 Time course of modifications after morphine challenge in the phosphorylation levels of DARPP-32, GluR1 and NR1 in the CPu of control and morphine-sensitized rats. Levels of phospho-Thr75 DAR- PP-32 (a), phospho-Thr34 DARPP-32 (b), phospho-Ser845 GluR1 (c) and phospho-Ser897 NR1 (d) were examined using phosphorylation state-specific antibodies. Levels in the CTR + MF group are expressed as mean ± SEM percentage of the levels obtained in con- trol rats that did not receive acute morphine administration. Levels in the MF + MF group are expressed as mean ± SEM percentage of the levels obtained in morphine-sensitized rats that did not receive acute morphine administration. Analysis of the data with the Kruskal–Wallis test indicated a significant difference between groups (KW ¼ 19.82,

Article Snippet: Immunoblotting was carried out with phosphorylation state-specific antibodies against phospho-Thr34 DARPP32 and phospho-Thr75 DARPP-32 (Cell Signalling Technology, Beverly, MA, USA), phospho-Ser845 GluR1 and phospho-Ser897 NR1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or with nonphosphorylation state-specific antibodies against total DARPP-32 (Cell Signalling Technology), total GluR1 and NR1 (Santa Cruz Biotechnology) and Cdk5 (Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Control

Journal: Journal of neurochemistry

Article Title: Dopamine and cyclic AMP-regulated phosphoprotein-32 phosphorylation pattern in cocaine and morphine-sensitized rats.

doi: 10.1111/j.1471-4159.2004.02510.x

Figure Lengend Snippet: Fig. 3 Time course of modifications after morphine challenge in the phosphorylation levels of DARPP-32, GluR1 and NR1 in the NAc of control and morphine-sensitized rats. Levels of phospho-Thr75 DAR- PP-32 (a), phospho-Thr34 DARPP-32 (b), phospho-Ser845 GluR1 (c) and phospho-Ser897 NR1 (d) were examined using phosphorylation state-specific antibodies. Levels in the CTR + MF group are ex- pressed as mean ± SEM percentage of the levels obtained in control rats that did not receive acute morphine administration. Levels in the MF + MF group are expressed as mean ± SEM percentage of the levels obtained in morphine-sensitized rats that did not receive acute morphine administration. Analysis of the data with the Kruskal–Wallis test indicated a significant difference between groups (KW ¼ 23.08,

Article Snippet: Immunoblotting was carried out with phosphorylation state-specific antibodies against phospho-Thr34 DARPP32 and phospho-Thr75 DARPP-32 (Cell Signalling Technology, Beverly, MA, USA), phospho-Ser845 GluR1 and phospho-Ser897 NR1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or with nonphosphorylation state-specific antibodies against total DARPP-32 (Cell Signalling Technology), total GluR1 and NR1 (Santa Cruz Biotechnology) and Cdk5 (Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Beneficial Effect of a Selective Adenosine A 2A Receptor Antagonist in the APPswe/PS1dE9 Mouse Model of Alzheimer’s Disease

doi: 10.3389/fnmol.2018.00235

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: Anti-phospho-GluR1 (Ser845) , pSer845 , Mono , Rabbit , Merck Millipore , 1/1000.

Techniques:

Journal: Cells

Article Title: The Intranigral Infusion of Human-Alpha Synuclein Oligomers Induces a Cognitive Impairment in Rats Associated with Changes in Neuronal Firing and Neuroinflammation in the Anterior Cingulate Cortex

doi: 10.3390/cells11172628

Figure Lengend Snippet: Cortical GluR1 expression is decreased following H-αSynOs oligomers infusion . ( A ) GluR1 expression levels in the ACC (n = 50 subfields, n = 6 animals per group; ** p < 0.01 by Mann–Whitney test). Representative images of GluR1 (green) expression in the ACC of ( A 1 ) vehicle-infused and ( A 2 ) H-αSynOs-infused rats. Magnification 63×. Scale bar: 20 μm.

Article Snippet: For immunofluorescence, after pre-incubation of hippocampal and ACC sections in a blocking solution, the following unconjugated primary antibodies were used for single or double immunolabeling: goat polyclonal anti Iba-1 (1:1000; Novus Biologicals, Littleton, CO, USA); rabbit polyclonal anti TNF-α (1:500, Novus Biologicals, Littleton, CO, USA); rabbit polyclonal anti GFAP (glial fibrillary acidic protein, 1:2500, Novus Biologicals, Littleton, CO, USA); mouse monoclonal anti GluR1 (1:200, Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, MANN-WHITNEY