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: bioRxiv

Article Title: Metaplastic priming enables non-ionotropic NMDA receptor-mediated synaptic depotentiation in the hippocampus

doi: 10.1101/2025.02.28.640846

Figure Lengend Snippet: ( A ) Representative confocal microscopy images of hippocampal slices following electrophysiology stained for NeuN (grey, AF-647), GluA1 (blue, AF-488), GluA2 (red, AF-594), and DAPI (green) and corresponding merged images for naïve, cLTP, sLTP, cDEP, and sDEP slices (maximum intensity projection). The dotted white line shown in the merged image represents the region of interest (stratum radiatum, SR) for each slice. Scalebar = 200 µm. ( B ) After normalization to naïve slices, the GluA1/GluA2 ratio in the SR was higher following cLTP compared to sLTP (unpaired Student’s t test, mean difference = 0.277 ± 0.084 times the naïve GluA1/GluA2 ratio, p = 0.022). Data are means ± SEM from 4 (sLTP) and 3 (cLTP) biological replicates. ( C ) No differences in the GluA1/GluA2 ratio were observed following cDEP compared to sDEP (unpaired Student’s t test, mean difference = −0.002 ± 0.186 times the naïve GluA1/GluA2 ratio, p = 0.993). Data are means ± SEM from 5 (sDEP) and 3 (cDEP) biological replicates. ( D ) Fluorescence intensity profiles normalized to the maximum fluorescence intensity as a function of distance along the SR (normalized to 1) for GluA1 (left) and GluA2 (right) in naïve slices. Data are means ± SEM from 10 biological replicates. ( E ) Normalized fluorescence intensity profiles for GluA1 (left) and GluA2 (right) in slices fixed after cLTP or sLTP. Data are means ± SEM from 4 (sLTP) and 3 (cLTP) biological replicates. Differences in GluA1 fluorescence intensity along the SR relative to the maxima were observed between cLTP and sLTP (mixed effects model, distance x LTP F(99, 494) = 1.11, p = 0.239; main effect of LTP type F(1,5) = 9.43, p = 0.028). No statistically significant differences in GluA2 distribution were observed between cLTP and sLTP (mixed effects model, distance x LTP F(99, 494) = 1.23, p = 0.081, LTP type F(1, 5) = 3.11, p = 0.138). ( F ) Normalized fluorescence intensity profiles for GluA1 (left) and GluA2 (right) in slices fixed after cDEP or sDEP. Data are means ± SEM from 5 (sDEP) and 3 (cDEP) biological replicates. No statistically significant differences were observed between cDEP and sDEP for GluA1 (distance x DEP F(99, 591) = 0.459, p > 0.999, DEP type F(1, 6) = 2.07, p = 0.200) or GluA2 (distance x DEP F(99, 591) = 0.680, p = 0.991, DEP type F(1, 6) = 1.69, p = 0.242) using mixed effects analysis. Data from male (closed circles) and female (open circles) mice combined, 1-2 technical replicates per biological replicate.

Article Snippet: Primary antibodies used include anti-GluA1 (1:400, guinea pig host, Alomone labs, Jerusalem, Israel, cat nr #AGC-004-GP, RRID: AB_2340961), anti-GluA2 (1:1000, rabbit host, Abcam, cat nr #ab206293, RRID: AB_2800401), and NeuN (1:1000, mouse host, Millipore-Sigma, Burlington, MA, United States, cat nr #MAB377, RRID: AB_2298772).

Techniques: Confocal Microscopy, Staining, Fluorescence

Journal: bioRxiv

Article Title: Metaplastic priming enables non-ionotropic NMDA receptor-mediated synaptic depotentiation in the hippocampus

doi: 10.1101/2025.02.28.640846

Figure Lengend Snippet: ( A ) GluA1 normalized to naïve hippocampal slices following cDEP and sDEP in control conditions (black), in the presence of 100 µM 7-CK (purple) and in the presence of 50 µM APV (blue). DEP x treatment F(2, 15) = 0.240, p = 0.790. ( B ) GluA2 normalized to naïve slices following cDEP and sDEP in control conditions and in the presence of 7-CK or APV. DEP x treatment F(2, 15) = 0.220, p = 0.805. ( C ) pGluA1 S831/GluA1 ratio normalized to naïve hippocampal slices following cDEP and sDEP in control conditions and in the presence of 7-CK or APV. DEP x treatment F(2, 15) = 0.583, p = 0.571, main effect of DEP type F(1, 15) = 12.27, p = 0.003. Post-hoc cDEP+7-CK vs. sDEP+7-CK least squares (LS) mean difference = 1.3 ± 0.6 times naïve pGluA1 S831/GluA1 expression, p = 0.034; cDEP vs. sDEP LS mean difference = 1.4 ± 0.6 times naïve pGluA1 S831/GluA1 expression, p = 0.021. ( D ) pGluA1 S845/GluA1 ratio normalized to naïve hippocampal slices following cDEP and sDEP in control conditions and in the presence of 7-CK or APV. DEP x treatment F(2, 15) = 1.62, p = 0.231, main effect of DEP type (F(1, 15) = 5.88, p = 0.028) and drug treatment (F(2, 15) = 6.57, p = 0.0089). Post-hoc cDEP+7CK vs. cDEP+APV LS mean difference = 3.7 ± 1.4 times naïve pGluA1 S845/GluA1 expression, p = 0.054; sDEP vs. sDEP+7-CK LS mean difference = −4.4 ± 1.6 times naïve pGluA1 S845/GluA1 expression, p = 0.043; cDEP vs. sDEP LS mean difference = 4.3 ± 1.5 times naïve pGluA1 S845/GluA1 expression, p = 0.012. Data in (A-D) are means ± SEM from 4 (cDEP) and 3 (sDEP) biological replicates. ( E-H ) AMPAR expression and phosphorylation states are unaltered following cDEP in the presence of C1.1 compared to C1.1Scr. ( E ) GluA1 normalized to naïve hippocampal slices following cDEP in the presence of C1.1Scr (black) versus C1.1 (grey) (p = 0.555). ( F ) GluA2 normalized to naïve hippocampal slices following cDEP in the presence of C1.1Scr versus C1.1 (p = 0.876). ( G ) pGluA1 S831/GluA1 ratio normalized to naïve hippocampal slices following cDEP with C1.1Scr versus C1.1 (p = 0.238) ( H ) pGluA1 S845/GluA1 ratio normalized to naïve hippocampal slices following cDEP with C1.1Scr versus C1.1 (p = 0.067). Data in (E-G) are means ± SEM from 4 biological replicates per group. 1-2 technical replicates per biological replicate. Blots were cut to probe for each protein (see Fig. S5A-C). Each drug treatment condition was normalized to a naïve slice run in the same blot. Statistical comparisons were made using ordinary two-way ANOVA followed by Holm-Šídák post-hoc comparisons within and across drug treatment conditions (A-D) or unpaired Student’s t test (E-H) as appropriate.

Article Snippet: Primary antibodies used include anti-GluA1 (1:400, guinea pig host, Alomone labs, Jerusalem, Israel, cat nr #AGC-004-GP, RRID: AB_2340961), anti-GluA2 (1:1000, rabbit host, Abcam, cat nr #ab206293, RRID: AB_2800401), and NeuN (1:1000, mouse host, Millipore-Sigma, Burlington, MA, United States, cat nr #MAB377, RRID: AB_2298772).

Techniques: Control, Expressing

Journal: bioRxiv

Article Title: Metaplastic priming enables non-ionotropic NMDA receptor-mediated synaptic depotentiation in the hippocampus

doi: 10.1101/2025.02.28.640846

Figure Lengend Snippet: ( A ) Full blots stained for GluA1 (top left-hand side of blots), pGluA1 S831 (top right-hand side of blots) and GAPDH (bottom section of blots) after membranes were cut as indicated along dotted lines. Blots containing samples frozen after cDEP (left) and sDEP (right) are shown. Samples were treated during electrophysiology experiments with 7-CK or APV as indicated. ( B ) Full blots stained for GluA2 (top left-hand side of blots), pGluA1 S845 (top right-hand side of blots) and GAPDH (bottom of blots) after blots were cut along dotted lines. Blots containing samples frozen after cDEP (left) and sDEP (right) are shown with corresponding treatments indicated. ( C ) Full blots stained for GluA1 (top left-hand side of left blot), pGluA1 S831 (top right-hand side of left blot), GluA2 (top left-hand side of right blot) and pGluA1 S845 (top right-hand side of right blot). Cropped blots from main are indicated with the corresponding colour-coded boxes. Images of blots stained for pGluA1 S831 and S845 were mirrored in for clarity.

Article Snippet: Primary antibodies used include anti-GluA1 (1:400, guinea pig host, Alomone labs, Jerusalem, Israel, cat nr #AGC-004-GP, RRID: AB_2340961), anti-GluA2 (1:1000, rabbit host, Abcam, cat nr #ab206293, RRID: AB_2800401), and NeuN (1:1000, mouse host, Millipore-Sigma, Burlington, MA, United States, cat nr #MAB377, RRID: AB_2298772).

Techniques: Staining

Journal: bioRxiv

Article Title: Parkinson’s-linked LRRK2-G2019S derails AMPAR trafficking, mobility and composition in striatum with cell-type and subunit specificity

doi: 10.1101/2023.10.13.562231

Figure Lengend Snippet: ( A - C ) Surface biotinylation was used to isolate endogenous surface (s) GluA1 and sGluA2 in acute striatal slices derived from wildtype (WT) and Lrrk2 G2019S (GS) mice. A ) Representative Western blot images of sGluA1, sGluA2, and actin from dataset quantified in B and C . Scatterplot/bar graphs plot values normalized to WT ± SEM (n = 7 - 8 mice/genotype, 3 slices/mouse). Unpaired t test *p= 0.0492, compared to WT. D ) Confocal image of DARPP32-labeled (white) co-cultured SPN (left) and super-resolution, STED images (right) of dendritic processes. Punctate sGluA1 (magenta), sGluA2 (green) labeling (tagged prior to permeabilization) associates largely, but not completed with PSD95 labeling (blue). E and F ) Violin plots compare surface AMPAR area ( E ) and intensity ( F ) within masks defined by PSD95 labeling in SPNs. Unpaired t test, ****p<0.0001; ***p<0.0001; Mann Whitney test *p=0.04. n= 3 preps and 15 ROIs/genotype.

Article Snippet: Cultured neurons were live-labeled with a direct-conjugated, ATTO-594 (red) rabbit anti-GluA1 antibody (1:100, Alomone AGC-004-AR, RRID:AB_2340944) for 15 min at room temperature.

Techniques: Derivative Assay, Western Blot, Labeling, Cell Culture, MANN-WHITNEY

Journal: bioRxiv

Article Title: Parkinson’s-linked LRRK2-G2019S derails AMPAR trafficking, mobility and composition in striatum with cell-type and subunit specificity

doi: 10.1101/2023.10.13.562231

Figure Lengend Snippet: A ) Schematic outlines antibody feeding assay used to monitor GluA1 internalization in wildtype (WT) and Lrrk2 G2019S (GS) corticostriatal co-cultures (DIV16-18) and serves as a key for the colors used to show data. B and C ) Overlay images show labeled surface (s) GluA1 (green mask + red mask) and internalized (i) GluA1 (red mask only) signal contained within DARPP-32 labeled SPNs (shown at a reduced intensity to permit visualization of puncta. Masks were generated in Image J and magnification is shown in B. D - G ) Intensity distribution of green and red labeling along a 60 µm line scan. H ) Quantification of the internalization index of GluA1 receptors in WT and GS SPNs at 0 and 60 min (n = 12 - 16 cells, 3 preps/genotype). Two-way ANOVA (F ( , ) = 9.809, p = 0.0029), post hoc Šidák test **p = 0.0049.

Article Snippet: Cultured neurons were live-labeled with a direct-conjugated, ATTO-594 (red) rabbit anti-GluA1 antibody (1:100, Alomone AGC-004-AR, RRID:AB_2340944) for 15 min at room temperature.

Techniques: Feeding Assay, Labeling, Generated

Journal: bioRxiv

Article Title: Parkinson’s-linked LRRK2-G2019S derails AMPAR trafficking, mobility and composition in striatum with cell-type and subunit specificity

doi: 10.1101/2023.10.13.562231

Figure Lengend Snippet: A, C ) Bar graph/scatterplots comparing functional contribution of CP-AMPARs using ratios of EPSCs evoked in the presence/absence of NASPM in D 1 R ( A; Drd1tdTom+ ) and D 2 R ( C; Drd1tdTom- ) SPNs in WT and GS mice (P70 - P90) in acute slices through dorsal striatum. Bars are mean ± SEM (n = 11 cells, 5 - 6 mice/group, unpaired t test *p = 0.0122). Example traces ( B ) show AMPAR currents before (black) and after (lavender) bath application of NASPM (200µM, 10 min). D - G , Superresolution (tau-STED) images ( D, E ) and quantification ( F, G ) of synaptic and extrasynaptic receptors in 21 DIV WT and GS D 1 R SPNs expressing tdTomato (white, Drd1 Cre/+; Ai14 , in D, E, and used to segment D1R SPNs for F, G) and co-cultured with unlabeled cortical neurons of the same genotype. sGluA1 ( D , magenta, STED) and sGluA2 ( E , green, STED) puncta in relation to PSD95 labeled postsynaptic sites (blue, confocal). Circled zones are enlarged in the center panels. F, G ) Bar graph/scatterplots show synaptic ( F , within a mask defined by PSD95) and extrasynaptic ( G , outside a PSD95 mask) sGluA1 (lavender) and sGluA2 (green) nanoclusters in D 1 R SPNs. F : Unpaired t test **p = 0.0369; n = 16 ROIs/genotype). G : Unpaired t test ****p<0.0001; n = 16 ROIs/genotype. H - K ) Examples ( H ) and quantification ( I - K ) of FRAP experiments. (H) Time lapse confocal images pre- and postphotobleaching (dotted circles approximate ROIs) in WT and GS D 1 R SPNs labeled and co-cultured as above. Table ( I ) compares time constant, diffusion (D) and % mobile receptors. D was calculated using: D = 0.25 (r 2 /τ 1/2 ), where r refers to the bleach radius and τ 1/2 to the time constant 46 . Graph ( J ) plots normalized SEP-GluA1 fluorescence recov-ery in WT and GS neurons imaged every 2.5 s. Lighter shading is ± SEM. Two-way RM-ANOVA (F(89, 3115) = 17.79, **p < 0.001, n = 19-20 spines/genotype. Scatterplot ( K ) compares recovery of SEP-GluA1 intensity at T222.5 s time point relative to post-bleach in WT and GS D 1 R SPNs. One-way ANOVA (F(3, 70) = 24.15, p < 0.0001), post hoc Tukey’s multiple comparison test ****p<0.0001.

Article Snippet: Cultured neurons were live-labeled with a direct-conjugated, ATTO-594 (red) rabbit anti-GluA1 antibody (1:100, Alomone AGC-004-AR, RRID:AB_2340944) for 15 min at room temperature.

Techniques: Functional Assay, Expressing, Cell Culture, Labeling, Diffusion-based Assay, Fluorescence, Comparison

Journal:

Article Title: Regulation of GluR1 abundance in murine hippocampal neurones by serum- and glucocorticoid-inducible kinase 3

doi: 10.1113/jphysiol.2004.079582

Figure Lengend Snippet: A, representative Western blot. Glycosylated plasma membrane proteins expressed in oocytes were labelled with biotinyl-ConA. Oocytes were homogenized and plasma membrane proteins were streptavidin-precipitated. Samples including controls from uninjected oocytes were separated on a SDS gel, Western-blotted and probed with a primary rabbit immunoaffinity-purified anti-GluR1 antibody. GluR1 protein has an apparent molecular mass of ∼105 kDa. P, plasma membrane protein (n = 18); SN, supernatant fraction containing intracellular protein (n = 1); T, total protein (n = 1). B, bar graph showing relative abundance of GluR1 plasma membrane protein. The band intensity was quantified by arithmetric analysis using the software Scion image. The values of three different blots from different batches were used for the statistical analysis. Due to the saturating conditions of the Western Blot procedure, the analysis represents only an estimation of relative abundance of GluR1 plasma membrane protein.

Article Snippet: For the detection of GluR1, primary rabbit immunoaffinity-purified anti-GluR1 antibody (1 μg μl −1 ; Upstate, Biomol, Hamburg, Germany) and secondary horseradish peroxidase-conjugated donkey anti-rabbit antibody (1:1000 dilution; Amersham Biosciences, Freiburg, Germany) were used.

Techniques: Western Blot, SDS-Gel, Purification, Software

Journal:

Article Title: Regulation of GluR1 abundance in murine hippocampal neurones by serum- and glucocorticoid-inducible kinase 3

doi: 10.1113/jphysiol.2004.079582

Figure Lengend Snippet: A, representative current traces measured in Xenopus oocytes in response to superfusion with 300 μm glutamate. All currents were measured at −70 mV. Horizontal scale bars indicate 5 s, and vertical scale bars represent 1 μA. B, GluR1 current amplitudes in oocytes expressing GluR1(L479Y) + DEPC-H2O, GluR1(L479Y) + SGK1, GluR1(L479Y) + SGK2, GluR1(L479Y) + SGK3 and GluR1(L479Y) + PKB normalized to the GluR1(L479Y) + DEPC-H2O currents. Numbers of oocytes are shown in parenthesis, and significant changes (P < 0.001) are indicated by ***.

Article Snippet: For the detection of GluR1, primary rabbit immunoaffinity-purified anti-GluR1 antibody (1 μg μl −1 ; Upstate, Biomol, Hamburg, Germany) and secondary horseradish peroxidase-conjugated donkey anti-rabbit antibody (1:1000 dilution; Amersham Biosciences, Freiburg, Germany) were used.

Techniques: Expressing

Journal:

Article Title: Regulation of GluR1 abundance in murine hippocampal neurones by serum- and glucocorticoid-inducible kinase 3

doi: 10.1113/jphysiol.2004.079582

Figure Lengend Snippet: A, original, representative Western blot of mouse hippocampal tissue. Immunostaining of protein fractions of hippocampal tissue of wild-type mice and SGK3-knockout mice using an immunoaffinity-purified rabbit anti-GluR1 antibody. GluR1 receptor expression was less pronounced in Sgk3−/− than in Sgk3+/+ hippocampal tissue. The same blot was stripped and reprobed with monoclonal anti-β-tubulin antibody. β-(ubulin protein expression was tested as a control, and was comparable in SGK3-knockout and wild-type mice. B, bar graph showing arithmetic means ± s.e.m. of relative abundance of GluR1 protein. Protein was isolated from three wild-type mice and five SGK3-knockout mice. **Significant difference between Sgk3+/+ and Sgk3−/−(P < 0.01). Due to the saturating conditions of the Western blot the analysis represents only an estimation of relative abundance of GluR1 plasma membrane protein.

Article Snippet: For the detection of GluR1, primary rabbit immunoaffinity-purified anti-GluR1 antibody (1 μg μl −1 ; Upstate, Biomol, Hamburg, Germany) and secondary horseradish peroxidase-conjugated donkey anti-rabbit antibody (1:1000 dilution; Amersham Biosciences, Freiburg, Germany) were used.

Techniques: Western Blot, Immunostaining, Knock-Out, Purification, Expressing, Isolation