Journal: Biochimica et biophysica acta

Article Title: The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells

doi: 10.1016/j.bbamcr.2013.03.021

Figure Lengend Snippet: Cerebellar granule cells at 5 DIV were transfected with the cGKII siRNA and immunostained at 8 DIV with the specific rabbit NT-GluA1 antiserum and a mouse anti-MAP2 antibody. (A) Representative images of GluA1/MAP2 immunolabelling in control (scrambled siRNA) and cGKII silenced cells. (B and E) Cumulative probability plots and bar chart for the GluA1/MAP2 mean fluorescence intensity (**p<0.001, Mann–Whitney and Kolmogorov–Smirnov tests): Control 878 ROIs 0.27±0.07; scrambled siRNA 3,365 ROIs 0.26±0.03; cGKII siRNA 2,716 ROIs 0.06±0.01. (D) Cumulative probability plots of the mean MAP2 fluorescence intensity (a.u.) of cells also labelled with anti-GluA1 (MAP2 mean intensity 60.7±0.54, 58.7±1, 59.2±1.5 for control, scrambled siRNA and cGKII siRNA respectively: p>0.05, Mann–Whitney and Kolmogorov–Smirnov tests).

Article Snippet: The cell surface GluA1 receptors were labelled by incubating live cultures for 20 min at 37 °C with rabbit NT-GluA1 (20 μg/ml: Calbiochem, Millipore Ibérica, Madrid, Spain) in HBM solution as described previously [ 32 ].

Techniques: Transfection, Fluorescence, MANN-WHITNEY

Journal: Biochimica et biophysica acta

Article Title: The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells

doi: 10.1016/j.bbamcr.2013.03.021

Figure Lengend Snippet: (A) Cerebellar granule cells maintained for 8 DIV were subjected to different treatments as shown: incubation with 8Br-cGMP (500 μM) for 20 minutes or inhibition with KT5823 during 30 minutes followed by incubation with a GluA1-NT antibody (20 μg/ml) for 20 minutes as indicated in the Methods section. Images represent the immunostaining for synapsin I and GluA1, the merged image and an amplification of each condition. (B) Density of GluA1 clusters in dendrites (clusters/μm). ROI’s of the specific antibody were selected for each condition from at least 15 fields from 2 different experiments. (C) Analysis of the fraction of GluA1 clusters apposed to synapsin I puncta, either upon stimulation or inhibition (n=30 fields from 2 independent experiments). (D) Cumulative probability plots for GluA1 mean fluorescence intensity (mean values: control 19.4±0.06; 8Br-cGMP 23.1±0.11; KT5823 16.4±0.03; 8Br-cGMP+KT 16.7±0.06, a.u.). In B and C the data are represented as the mean ± SEM and analyzed with an unpaired, two-sample t test (*p<0.001). The Komolgorov-Smirnov test was used for the cumulative probability. Scale bars: 10 μm.

Article Snippet: The cell surface GluA1 receptors were labelled by incubating live cultures for 20 min at 37 °C with rabbit NT-GluA1 (20 μg/ml: Calbiochem, Millipore Ibérica, Madrid, Spain) in HBM solution as described previously [ 32 ].

Techniques: Incubation, Inhibition, Immunostaining, Amplification, Fluorescence

Journal: Biochimica et biophysica acta

Article Title: The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells

doi: 10.1016/j.bbamcr.2013.03.021

Figure Lengend Snippet: (A) Cerebellar granule cells maintained for 8 DIV were treated for 10 minutes with NMDA (50 μM) + glycine (10μM), or for 30 minutes with KT5823, followed by incubation with a GluA1-NT antibody (20 μg/ml) over 20 minutes. The images represent the immunostaining of synapsin I and GluA1, the merged image and an amplification of each condition. (B) The density of GluA1 clusters in dendrites (clusters/μm), selecting ROI’s of the specific antibody from at least 15 fields from 2 different experiments for each condition. (C) Analysis of the fraction of GluA1 clusters apposed to synapsin I puncta, either upon stimulation or inhibition (n=30 fields from two independent experiments with different cell cultures). (D) Cumulative probability plots of GluA1 mean fluorescence intensity (mean values: control 19.7±0.04; 8Br-cGMP 23.4±0.08; KT5823 16.2±0.06; 8Br-cGMP+KT 16.9±0.07, a.u.). In B and C, the data are represented as the mean ± SEM and analyzed using unpaired, two-sample t test (*p<0.001). For the cumulative probability the Komolgorov-Smirnov test was used. Scale bars: 10 μm.

Article Snippet: The cell surface GluA1 receptors were labelled by incubating live cultures for 20 min at 37 °C with rabbit NT-GluA1 (20 μg/ml: Calbiochem, Millipore Ibérica, Madrid, Spain) in HBM solution as described previously [ 32 ].

Techniques: Incubation, Immunostaining, Amplification, Inhibition, Fluorescence

Journal: Biochimica et biophysica acta

Article Title: The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells

doi: 10.1016/j.bbamcr.2013.03.021

Figure Lengend Snippet: (A) Expression of GluA1 (mRNA and protein) during the development of cerebellar granule cells in culture. Total RNA was extracted from freshly isolated cells (FIC), and cells 7 and 14 DIV. Equal amounts of mRNA were used for Reverse Transcription and then real time PCR reactions were performed with the specific primers described in the Methods. The results correspond to the mean ± SEM of four experiments performed in triplicate and they are expressed as 50-ΔCT. Total protein extracts (15 μg) obtained from cells 3, 7 and 14 DIV were resolved by electrophoresis and transferred to nitrocellulose membranes, which were probed with a rabbit polyclonal anti-GluA1 N-terminal specific antiserum. (B and C) Cells were incubated with the compounds indicated (see the Methods section: NMDA implies NMDA 50 μM + glycine 10 μM). Total GluA1 was assayed with a rabbit anti-GluA1 antiserum, phosphorylated GluA1 at S845 was assayed with a rabbit PO-S845-GluA1 antiserum and β-tubulin was used as a loading control. (D and E) The diagrams show the quantification of several experiments, where the phospho-specific signal was normalized to the total amount of GluA1 and the final values were normalized to the controls.(D, Control: 1; 8Br-cGMP: 1.30±0.03; KT5823: 0.79±0.04; 8Br-cGMP+KT: 0.75±0.08; E, Control: 1; NMDA: 1.24±0.02; KT5823: 0.63±0.05; NMDA+KT: 0.82±0.09). The data are represented as the mean ± SEM and analyzed with an unpaired, two-sample t test (*p < 0.05, n = 7).

Article Snippet: The cell surface GluA1 receptors were labelled by incubating live cultures for 20 min at 37 °C with rabbit NT-GluA1 (20 μg/ml: Calbiochem, Millipore Ibérica, Madrid, Spain) in HBM solution as described previously [ 32 ].

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Electrophoresis, Incubation

Journal: Biochimica et biophysica acta

Article Title: The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells

doi: 10.1016/j.bbamcr.2013.03.021

Figure Lengend Snippet: Subcellular fractions of the cerebellum from 14 day old WT and cGKII−/− mice were analyzed in immunoblots. P2: crude membranes; syn: synaptoneurosome; PSD: postsynaptic density. (B) Quantification of GluA1 immunoreactivity normalized to β-actin and finally, to WT values (*p < 0.05, n = 3). (C) Cerebellar fractions probed in immunoblots with a rabbit PO-S845-GluA1 antiserum. (D) Quantification of GluA1 phosphorylation in the synaptoneurosomal and crude PSD fractions. (E). Ratio between the phosphorylated and total GluA1 in the synaptic fractions. Data are represented as the mean ± SEM and analyzed using unpaired, two-sample t test (n=3; *p<0.05).

Article Snippet: The cell surface GluA1 receptors were labelled by incubating live cultures for 20 min at 37 °C with rabbit NT-GluA1 (20 μg/ml: Calbiochem, Millipore Ibérica, Madrid, Spain) in HBM solution as described previously [ 32 ].

Techniques: Western Blot

Journal: Biochimica et biophysica acta

Article Title: The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells

doi: 10.1016/j.bbamcr.2013.03.021

Figure Lengend Snippet: Solubilized extracts of the rat (P14) cerebellum were immunoprecipitated using a mouse anti-flag antibody (2 μg, lane 1), rabbit anti-cGKII polyclonal antiserum (2 μg, lane 2), rabbit anti-HA antiserum (2 μg, lane3), a rabbit anti-GluA1 polyclonal antiserum (2 μg, lane 4) or a mouse anti-GluA1 monoclonal antibody (2mg, lane 4 of cGKII IB). Extracts (Crude) and immunoprecipitates (IP) were analyzed in immunoblots probed with a rabbit anti-GluA1 polyclonal antiserum (1 μg/ml) and rabbit anti-cGKII (1 μg/ml) antiserum. HRP-conjugated anti-rabbit IgG and anti-mouse TrueBlot™ (1:1000) antibodies were used as secondary antibodies to avoid IgG cross-reactivity. The immunoreactive bands were visualized by chemiluminescence.

Article Snippet: The cell surface GluA1 receptors were labelled by incubating live cultures for 20 min at 37 °C with rabbit NT-GluA1 (20 μg/ml: Calbiochem, Millipore Ibérica, Madrid, Spain) in HBM solution as described previously [ 32 ].

Techniques: Immunoprecipitation, Western Blot

Journal: Frontiers in Synaptic Neuroscience

Article Title: Correlative Assembly of Subsynaptic Nanoscale Organizations During Development

doi: 10.3389/fnsyn.2022.748184

Figure Lengend Snippet: Synapse volumes increase correlatively during synaptic maturation. (A–D) Representative distribution of RIM1/2 and GluA1 under stochastic optical reconstruction microscopy (STORM). Scale 2 μm in top panels and 500 nm in lower panels. (E–G) Volumes of identified synaptic RIM1/2, GluA1, and PSD-95 clusters across different developmental stages. Numbers in bars denote the synapse numbers. (H,I) Correlations between the volumes of GluA1 and RIM1/2 clusters (H) and the volumes of PSD-95 and RIM1/2 (I) within the same synapses. Linear regressions were conducted on synapses of DIV7 (gray circles and line) and DIV18 (dark blue circles and line). Data from synapses of DIV10 and 14 were plotted with dark yellow and dark red crosses. Also refer to , and for more details on correlations and statistics. All experiments were repeated ≥3 times.

Article Snippet: For co-staining of GluA1 and RIM1/2, as both antibodies were from rabbits, staining was performed separately and the first primary antibody (GluA1) was blocked and converted with a goat anti-rabbit Fab fragment (JacksonImmuno 111-007-003) for 2 h at RT, and then recognized by secondary antibodies.

Techniques: Microscopy

Journal: Frontiers in Synaptic Neuroscience

Article Title: Correlative Assembly of Subsynaptic Nanoscale Organizations During Development

doi: 10.3389/fnsyn.2022.748184

Figure Lengend Snippet: The heterogeneity of synaptic protein distribution increases with development. (A–D) Representative density maps of synaptic GluA1 at different developmental stages. Scale bars, 200 nm. (E–G) Normalized autocorrelation functions of RIM1/2 (E) , GluA1 (F) , and PSD-95 (G) . g a above 1 suggests a significant non-uniform distribution. (H–J) Developmental changes of nanocluster number (left), normalized density within nanocluster (middle), and nanocluster volume of RIM1/2 (H) , GluA1 (I) , and PSD-95 (J) . Numbers in bars denote the synapse numbers. Also refer to for more statistical details. All experiments were repeated ≥3 times.

Article Snippet: For co-staining of GluA1 and RIM1/2, as both antibodies were from rabbits, staining was performed separately and the first primary antibody (GluA1) was blocked and converted with a goat anti-rabbit Fab fragment (JacksonImmuno 111-007-003) for 2 h at RT, and then recognized by secondary antibodies.

Techniques:

Journal: Frontiers in Synaptic Neuroscience

Article Title: Correlative Assembly of Subsynaptic Nanoscale Organizations During Development

doi: 10.3389/fnsyn.2022.748184

Figure Lengend Snippet: Correlation between presynaptic and postsynaptic protein heterogeneity in mature synapses. (A,B) Scatter plots for heterogeneity of GluA1 (A) and PSD-95 (B) against that of RIM1/2. All data points across all developmental stages could be fitted with linear functions as shown with lines. (C,D) Linear regressions of the relationships between heterogeneity of GluA1/PSD-95 and RIM1/2 at DIV7 (gray) and DIV18 (dark blue). (E) Relationship between heterogeneity and cluster volume of RIM1/2 at DIV7 (gray) and DIV18 (dark blue). Data points with cluster volume >1 × 10 7 nm 3 were fitted with linear functions. It is noted that g a of immature synapses was significantly lower than that of matured synapses. (F) Averaged g a of synapses with cluster volume of 1–4 × 10 7 nm 3 for immature (DIV7-10) and mature synapses (DIV18). Also refer to for more details. *** p < 0.001, t-test. All experiments were repeated ≥3 times.

Article Snippet: For co-staining of GluA1 and RIM1/2, as both antibodies were from rabbits, staining was performed separately and the first primary antibody (GluA1) was blocked and converted with a goat anti-rabbit Fab fragment (JacksonImmuno 111-007-003) for 2 h at RT, and then recognized by secondary antibodies.

Techniques:

Journal: Frontiers in Synaptic Neuroscience

Article Title: Correlative Assembly of Subsynaptic Nanoscale Organizations During Development

doi: 10.3389/fnsyn.2022.748184

Figure Lengend Snippet: Evolvement of trans-synaptic nano-alignment during synaptic maturation. (A–D) Representative examples of synapses with RIM1/2 (red) and GluA1 (blue) co-labeled and imaged with STORM. Thick color denotes detected nanoclusters. Scale bar, 200 nm. (E) Normalized local density of GluA1 along with distances from RIM1/2 nanoclusters for synapses at different developmental stages. (F) , Averaged enrichment of GluA1 within 50 nm from peaks of RIM1/2 nanoclusters ( n = 85, 60, 30, 54, and 54 nanoclusters). The open bar represents the enrichment indices of synapses with the position of nanoclusters randomized within synaptic clusters. (G) Fraction of nanoclusters that were enriched with protein across the cleft. (H–J) Enrichment between RIM1/2 and PSD-95 for synapses at different developmental stages. * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA with Tukey's multiple comparisons test in (F,I) , z -test in (G,J) . Also refer to for more statistical details. All experiments were repeated ≥3 times.

Article Snippet: For co-staining of GluA1 and RIM1/2, as both antibodies were from rabbits, staining was performed separately and the first primary antibody (GluA1) was blocked and converted with a goat anti-rabbit Fab fragment (JacksonImmuno 111-007-003) for 2 h at RT, and then recognized by secondary antibodies.

Techniques: Labeling

Journal: Brain sciences

Article Title: Arc-Mediated Synaptic Plasticity Regulates Cognitive Function in a Migraine Mouse Model.

doi: 10.3390/brainsci13020331

Figure Lengend Snippet: Figure 4. Determination of hippocampal protein in mice. **: p < 0.01, *: p < 0.05. (A): WB results of hippocampal protein. (B): NR2B/GAPDH ratio. (C): Arc/GAPDH ratio. (D): GluR1/GAPDH ratio. (E): SYP/β-Actin ratio.

Article Snippet: Then, the sections were closed and incubated overnight with the following primary antibody: mice anti-mice Arc (1:100; Santa Cruz, USA), mice anti-mice GluR1 (1:100; Santa Cruz, USA), and rabbit anti-mice SYP (1:100, Wanleibio, China).

Techniques:

Journal: Brain sciences

Article Title: Arc-Mediated Synaptic Plasticity Regulates Cognitive Function in a Migraine Mouse Model.

doi: 10.3390/brainsci13020331

Figure Lengend Snippet: Figure 5. Determination of protein in the prefrontal cortex and hippocampus. ***: p < 0.001, **: p < 0.01, *: p < 0.05. (A): WB results of prefrontal cortex protein. (B): NR2B/GAPDH ratio. (C): GluR1/GAPDH ratio. (D): Arc/GAPDH ratio. (E): WB results of hippocampus protein. (F): NR2B/GAPDH ratio. (G): GluR1/GAPDH ratio. (H): Arc/GAPDH ratio.

Article Snippet: Then, the sections were closed and incubated overnight with the following primary antibody: mice anti-mice Arc (1:100; Santa Cruz, USA), mice anti-mice GluR1 (1:100; Santa Cruz, USA), and rabbit anti-mice SYP (1:100, Wanleibio, China).

Techniques:

Journal: Brain sciences

Article Title: Arc-Mediated Synaptic Plasticity Regulates Cognitive Function in a Migraine Mouse Model.

doi: 10.3390/brainsci13020331

Figure Lengend Snippet: Figure 9. Immunofluorescence detection of GluR1 protein in the prefrontal cortex and hippocampal CA1 region. ***: p < 0.001. The blue light point in the figure is the nucleus. The red light dot is GluR1, and the bottom left corner is a zoomed-in view of it. (A): GluR1 protein in the prefrontal cortex of the IS+S. (B): GluR1 protein in the prefrontal cortex of the IS. (C): GluR1 protein in the prefrontal cortex of the IS+M. (D): GluR1 protein in the prefrontal cortex of the Control. (E): Comparison of GluR1’s mean optical density in each group’s prefrontal cortex. (F): GluR1 expression in the hippocampal CA1 region of the IS+S. (G): GluR1 expression in the hippocampal CA1 region of the IS. (H): GluR1 expression in the hippocampal CA1 region of the IS+M. (I): GluR1 expression in the hippocampal CA1 region of the Control. (J): Comparison of GluR1’s mean optical density in each group’s hippocampal CA1 region.

Article Snippet: Then, the sections were closed and incubated overnight with the following primary antibody: mice anti-mice Arc (1:100; Santa Cruz, USA), mice anti-mice GluR1 (1:100; Santa Cruz, USA), and rabbit anti-mice SYP (1:100, Wanleibio, China).

Techniques: Control, Comparison, Expressing

Journal: Brain sciences

Article Title: Arc-Mediated Synaptic Plasticity Regulates Cognitive Function in a Migraine Mouse Model.

doi: 10.3390/brainsci13020331

Figure Lengend Snippet: Figure 10. Cellular protein expression. NGF: nerve growth factor group, n = 4; Control: Control group, n = 4; N + M: NGF + memantine group, n = 4. ***: p < 0.001, **: p < 0.01, *: p < 0.05. (A): Western blot results of cultured cells. (B): The ratio of NR2B, NR1, Arc, GluR1, SYP to β-Actin, respectively.

Article Snippet: Then, the sections were closed and incubated overnight with the following primary antibody: mice anti-mice Arc (1:100; Santa Cruz, USA), mice anti-mice GluR1 (1:100; Santa Cruz, USA), and rabbit anti-mice SYP (1:100, Wanleibio, China).

Techniques: Expressing, Control, Western Blot, Cell Culture