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Figure Lengend Snippet: a Rph3A droplets condensed with the CTD of GluN2A in the optoDroplet assay. b Magnified images, in which the lines indicate the fluorescence intensity profiles of condensates in the white squares in a. Scale bar, 1 μm. c Representative images and quantification of fluorescence recovery from the FRAP analysis of GluN2A-CTD condensed with Rph3A droplets. The data are displayed as the mean ± SEM ( n = 6 puncta). d Fusion events of GluN2A-CTD condensed with Rph3A droplets in HEK293 cells stimulated with blue light. The image of cell at time point 0 s (left) and the magnified time-lapse images of the white squares in left (right) are showed. The large punctum (white arrow) is occasional preactivation. See also Supplementary Fig. . e The recombinant GluN2A-CTD mCherry fusion protein formed droplets with Rph3A-EGFP at a concentration of 10 μM in buffer containing 2% PEG8000 (top) but showed a diffuse distribution in buffer without Rph3A (bottom). The mCherry protein diffused in the presence of Rph3A-EGFP at the same concentration (middle). f Representative images and quantification of fluorescence recovery from the FRAP analysis of droplets composed of GluN2A-CTD in the presence of Rph3A. The data are displayed as the mean ± SEM ( n = 8 droplets). Scale bar, 1 μm. g Fusion events of droplets composed of GluN2A-CTD in the presence of Rph3A. Time-lapse images of fusing droplets are showed. h Droplets of Rph3A containing IDR2 condensed with GluN2A-CTD. The big punctum (white arrow) is occasional preactivation foci. See also Supplementary Fig. . i Magnified images, in which the lines indicate the fluorescence intensity profiles of condensates in the white squares in h. Scale bar, 1 μm. Source data of c and f are provided in the Source Data file.

Article Snippet: Cultured neurons were washed with HBS 2 times and incubated with an anti-GluN2A/GluN2B antibody (Alomone AGC-002/AGC-003, 1:200 dilution) for 20 min at 37 °C in HBS buffer.

Techniques: Fluorescence, Recombinant, Concentration Assay

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Figure Lengend Snippet: a Representative images of HEK293 cells transfected with different combinations of PSD95, GluN2A-CTD, WT, and R9A Rph3A. b Sizes of puncta composed of different combinations of the proteins in A. The data are displayed as the mean ± SEM (PSD95/GluN2A-CTD: n = 78 puncta; PSD95/GluN2A-CTD/Rph3A WT: n = 73 puncta; PSD95/GluN2A-CTD/Rph3A R9A: n = 66 puncta, ** p < 0.01, one-way ANOVA followed by Tukey’s multiple comparisons test). c Representative images of droplets formed by different combinations of PSD95, GluN2A-CTD, WT, and R9A Rph3A recombinant fusion proteins in a cell-free system. d Sizes of droplets composed of different combinations of proteins in c . The data are displayed as the mean ± SEM ( n = 73 droplets for each group, * p < 0.05, one-way ANOVA followed by Tukey’s multiple comparisons test). Source data and p values of b and d are provided in the Source Data file.

Article Snippet: Cultured neurons were washed with HBS 2 times and incubated with an anti-GluN2A/GluN2B antibody (Alomone AGC-002/AGC-003, 1:200 dilution) for 20 min at 37 °C in HBS buffer.

Techniques: Transfection, Recombinant

Journal:

Article Title:

doi:

Figure Lengend Snippet: a Representative images of EGFP-tagged WT and R9A Rph3A at dendrites of hippocampal neurons. Yellow triangles indicate synaptic puncta, and white triangles indicate extrasynaptic puncta of Rph3A. The density, size, and synaptic localization of the puncta of WT and R9A Rph3A, and the sizes of synaptic and extrasynaptic WT Rph3A puncta were also quantified. The data are displayed as the mean ± SEM (WT: n = 10 dendrites from 5 neurons; R9A: n = 6 dendrites from 3 neurons, ** p < 0.01, two-tailed unpaired t test). b Representative images and quantification of fluorescence recovery from the FRAP analysis of Rph3A puncta at the dendrites of hippocampal neurons. The data are displayed as the mean ± SEM ( n = 11 puncta). c Representative images from the FRAP assay of SEP-GluN2A in Rph3A-knockdown and Rph3A-reexpressing (WT and R9A) hippocampal neurons. d Quantification of the fluorescence recovery shown in c . The data are displayed as the mean ± SEM (Scramble: n = 17 puncta; Rph3A shRNA: 20 puncta; Rph3A shRNA-Rph3A WT: 18 puncta; Rph3A shRNA-Rph3A R9A: 20 puncta). e The recovery rate was calculated as the half-time of the recovery curve. The data are displayed as the mean ± SEM (n numbers are defined in d, * p < 0.05, one-way ANOVA followed by Dunnett’s multiple comparisons test). f The mobile fraction of GluN2A was calculated based on the plateau of the recovery curve. The data are displayed as the mean ± SEM (n numbers are defined in d, ** p < 0.01, one-way ANOVA followed by Dunnett’s multiple comparisons test). g Representative images of surface GluN2A and Homer1 staining in Rph3A-knockdown and Rph3A-reexpressing neurons. h-j Quantification of synaptic and extrasynaptic surface GluN2A cluster fluorescence intensity and density and the percentage of synaptic GluN2A clusters. The data are displayed as the mean ± SEM ( n = 10 dendrites from 5 neurons for each group, * p < 0.05, ** p < 0.01, one-way ANOVA followed by Dunnett’s multiple comparisons test (h, i) or by Tukey’s multiple comparisons test (j)). k Representative trace of NMDA eEPSCs in Rph3A-knockdown and Rph3A-reexpressing neurons. l Quantification of NMDA eEPSCs in Rph3A-knockdown and Rph3A-reexpressing neurons. The data are displayed as the mean ± SEM (Scramble: n = 58 neurons; Rph3A shRNA: 25 neurons; Rph3A shRNA-Rph3A WT: 22 neurons; Rph3A shRNA-Rph3A R9A: 19 neurons, * p < 0.05, one-way ANOVA followed by Dunnett’s multiple comparisons test). The full images of a , b , c , g are showed in Supplementary Fig. . Source data and p values of a , b , d , e , f , h , i , j and l are provided in the Source Data file.

Article Snippet: Cultured neurons were washed with HBS 2 times and incubated with an anti-GluN2A/GluN2B antibody (Alomone AGC-002/AGC-003, 1:200 dilution) for 20 min at 37 °C in HBS buffer.

Techniques: Two Tailed Test, Fluorescence, FRAP Assay, Knockdown, shRNA, Staining

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Figure Lengend Snippet: a Schematic illustration of dND-fused constructs used to restore phase separation of Rph3A. b OptoDroplet assay of indicated constructs in a. The number of puncta per cell was plotted. Data are displayed as the mean ± SEM (dND: n = 7 cells; dND-IDR1-R9A cells: n = 9 cells; IDR1-R9A: n = 6 cells). c Representative images of EGFP-tagged R9A and dND-fused Rph3A R9A at dendrites of hippocampus neurons. The density and synaptic localization of puncta were quantified. The data are displayed as the mean ± SEM (R9A: n = 6 dendrites from 3 neurons; dND-R9A: n = 9 dendrites from 5 neurons, ** p < 0.01, two-tailed unpaired t test). d Representative images of the FRAP assay of SEP-GluN2A in Rph3A-knockdown and Rph3A-reexpressing neurons. e Quantification of the fluorescence recovery in d. The data are displayed as the mean ± SEM (Rph3A WT: n = 9 puncta; Rph3A R9A: n = 9 puncta; dND-Rph3A R9A: n = 9 puncta; dND: n = 10 puncta). f The recovery rate of SEP-GluN2A. The data are displayed as the mean ± SEM (n numbers are defined in e, * p < 0.05, ** p < 0.01, one-way ANOVA followed by Tukey’s multiple comparisons test). g The mobile fraction of SEP-GluN2A. The data are displayed as the mean ± SEM (n numbers are defined in e, ** p < 0.01, one-way ANOVA followed by Tukey’s multiple comparisons test). h Representative images of surface GluN2A and Homer1 staining in Rph3A-knockdown and Rph3A-reexpressing neurons. i – k Quantification of the percentage of synaptic GluN2A clusters, synaptic and extra-synaptic surface GluN2A cluster fluorescence intensity and density. The data are displayed as the mean ± SEM ( n = 10 dendrites from 5 neurons for each group, * p < 0.05, ** p < 0.01, one-way ANOVA followed by Tukey’s multiple comparisons test). The full images of c , d and h are showed in Supplementary Fig. . Source data and p values of b , c , e , f , g , i , j and k are provided in the Source Data file.

Article Snippet: Cultured neurons were washed with HBS 2 times and incubated with an anti-GluN2A/GluN2B antibody (Alomone AGC-002/AGC-003, 1:200 dilution) for 20 min at 37 °C in HBS buffer.

Techniques: Construct, Two Tailed Test, FRAP Assay, Knockdown, Fluorescence, Staining

Journal:

Article Title:

doi:

Figure Lengend Snippet: 5-HT1b and 5-HT2a receptors are expressed in growth cones and filopodia. a–j Sensory neuron growth cones immunostained for serotonin receptors, 5-HT1b (green in a, red in g), 5-HT2a (green, d and h) as well as filamentous actin to highlight the extent of the growth cone (Phalloidin, red; b and e). The receptors 5-HT1b and 5HT2a were observed as a widespread punctate distribution throughout the growth cone (g–h). Punctate expression was evident in filopodia (j) with isolated 5-HT1b (green, *), 5-HT2a (red, #) as well as colocalised (arrowheads) puncta on filopodial shafts. There was prominent colocalization of 5-HT2a and 5-HT1b receptors in peripheral areas (i, arrow). Scale bars are 5 μm (a–i) and 1 μm (j). k Turning responses to serotonin were sensitive to chlorpromazine. Attractive turning of growth cones to 5HT-Lo (n = 7, p = <0.0001) and repulsion to 5HT-Hi (n = 8, p = 0.003) were significantly different when cultures were treated with chlorpromazine. l Axon extension was not significantly different in any experimental treatments (ns, p > 0.05). (Mann–Whitney u-test)

Article Snippet: Immunocytochemistry DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Expressing, Isolation, MANN-WHITNEY

Journal:

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Figure Lengend Snippet: 5HT-Lo elicits growth cone attraction through the 5-HT2a receptor. a Growth cone attraction was measured in the presence of 5HT-Lo gradients (p = <0.0001) and TCB-2 gradients (p = 0.0002). Growth cone attraction to 5HT-Lo was abolished by pharmacological application of the selective 5-HT2a receptor antagonist, ritanserin (n = 17, p = 0.0001) and the PLC inhibitor, U-73122 (n = 9, p = 0.0018). b Growth cone attraction to 5HT-Lo was not perturbed when growth cones treated with a specific 5-HT1b antagonist, GR55562 (n = 8, p = <0.0001), or the 5-HT3 antagonist ondansetron (n = 8, p = 0.0082). Depletion of ER calcium with thapsigargin (n = 19, p = 0.0003) abolished attractive turning to 5HT-Lo, while inhibition of VGCCs with nifedipine (n = 15, p = 0.0330) had no significant effect. Turning angles were compared to vehicle and 5HT-Lo and only significant differences shown in (a) and (b). Kruskal–Wallis, Dunn’s multiple comparison test. c, d Axon extension was not significantly different in any experimental treatments (ns, p > 0.05). Kruskal–Wallis, Dunn’s multiple comparison test

Article Snippet: Immunocytochemistry DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Inhibition, Comparison

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Figure Lengend Snippet: 5HT-Hi promotes growth cone repulsion through the 5-HT1b receptor. a Growth cone repulsion was measured in the presence of 5HT-Hi gradients (p = <0.0001) and CP-94253 gradients (p = 0.0018). Growth cone repulsion to 5HT-Hi was altered to levels indistinguishable from random growth (vehicle) with application of the selective antagonist to 5-HT1b, GR55562 (n = 16, p = <0.0001), by activating adenylate cyclase with forskolin (n = 8, p = 0.0003) and restoration of cAMP signals with Sp-cAMPs (n = 8, p = 0.0001). b Repulsion to 5HT-Hi was not perturbed when growth cones were treated with the 5-HT2a receptor antagonist ritanserin (n = 8, p = 0.002) or the 5-HT3 antagonist ondansetron (n = 10, p = 0.0003). Inhibition of ER calcium release with thapsigargin (n = 10, p = <0.0001) or calcium influx with nifedipine (n = 8, p = <0.0001) did not alter growth cone repulsion from 5HT-Hi. Turning angles were compared to vehicle and 5HT-Hi. c, d Axon extension was not perturbed by any pharmacological application (ns, p > 0.05). Turning angles were compared to vehicle and 5HT-Hi. (Kruskal–Wallis, Dunn’s multiple comparison test)

Article Snippet: Immunocytochemistry DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Inhibition, Hi-C, Comparison

Journal:

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Figure Lengend Snippet: 5HT-Lo exposure causes asymmetric distribution of 5-HT receptors to the turning or motile side of growth cones. Schematic a showing growth cone divided into “near” and “far” regions with respect to micropipet position (located on the upper left side). b–d Growth cones immunostained after turning in response to 5HT-Lo and stained for 5-HT2a (green, b), 5-HT1b (red, c) and merged in (d). Dotted line separates the near and far regions of growth cone (near vs far). (e) Representative image of filopodia with 5-HT2a (green), 5-HT1b (red) receptor puncta. (f) There was no significant near/far bias in the number of filopodia following exposure to 5HT-Lo (n = 8, p = 0.9004), 5HT-Hi (n = 13, p = 0.5450) or BDNF (n = 10, p = 0.7907) compared to vehicle. (Data not normally distributed: Kruskal–Wallis, Dunn’s multiple comparison test). g There were significantly more (n = 36, p = 0.0033) 5-HT2a puncta in filopodia on the near side of growth cones exposed to 5HT-Lo compared to all other treatments. h There was no bias in 5-HT1b puncta in filopodia of growth cones. (Mann–Whitney U-test). (i-j) When entire growth cones were analysed, i 5HT-2a (n = 9, p = 0.0006) and j 5HT-1b (n = 9, p = 0.0028) receptor translocation was significantly biased in growth cones exposed to 5HT-Lo. All ratios were compared to vehicle (Data normally distributed: Shapiro–Wilk. One-way ANOVA, Tukey’s multiple comparison test). Scale bars are 5 μm (Fig. 7b–d) and 1 μm (Fig. 7e)

Article Snippet: Immunocytochemistry DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Staining, Comparison, MANN-WHITNEY, Translocation Assay

Journal:

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Figure Lengend Snippet: 5HT-Lo exposure causes asymmetric distribution of membrane 5-HT2a receptors to the motile side of growth cones. a–h Representative growth cones exposed to vehicle and 5HT-Lo gradients (micropipette on upper left side) and stained for membrane localization of receptors 5-HT1b (green, a, e), 5-HT2a (green, c, g) and merged with actin, (red, b, d, f, h). Dotted line separates the near and far regions of the growth cone. i Increase magnification of representative image h to show the presence of 5-HT2a receptor membrane on most filopodia (*) of the growth cone (yellow *, insets). Scale bars are 5 μm. (j-k)There was no significant near/far bias in the amount of f-actin (/area) (j) and the number of filopodia (k) in growth cones exposed to vehicle and 5HT-Lo. Kruskal–Wallis, Dunn’s multiple comparison post hoc test. (l) There was no significant bias in the total percentage of filopodia containing 5HT2a and 5HT1b puncta (60%). One-way ANOVA followed by Tukey’s multiple comparison post hoc test. (m) There was no significant near/far bias in the amount of 5-HT2a and 5-HT1b puncta per filopodia. (n) Analysis of 5-HT receptor membrane localization showed translocation of 5-HT2a (n = 18, p = <0.0001) to the near side of growth cones exposed to 5HT-Lo while no significant translocation of 5-HT1b (n = 15, p = 0.346) was observed. (Mann–Whitney U-test)

Article Snippet: Immunocytochemistry DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Membrane, Staining, Comparison, Translocation Assay, MANN-WHITNEY

Journal: eLife

Article Title: Astrocyte GluN2C NMDA receptors control basal synaptic strengths of hippocampal CA1 pyramidal neurons in the stratum radiatum

doi: 10.7554/eLife.70818

Figure Lengend Snippet:

Article Snippet: antibody , Anti-GluN2A/B (Rabbit polyclonal) , Synaptic Systems , CAT# 244003 RRID: AB_10804284 , (WB 1:1000) (IP 5 μg / 40 μl ProteinA/G resin).

Techniques: Derivative Assay, Sequencing, Bicinchoninic Acid Protein Assay, Software, Staining, Fluorescence, Microscopy, Laser-Scanning Microscopy

Journal: Cells

Article Title: Flaxseed Oil Alleviates Trimethyltin-Induced Cell Injury and Inhibits the Pro-Inflammatory Activation of Astrocytes in the Hippocampus of Female Rats

doi: 10.3390/cells13141184

Figure Lengend Snippet: List of primary antibodies.

Article Snippet: Anti- NMDA Receptor 2A (GluN2A) , Merck Millipore (Burlington, Massachusetts, USA), rabbit monoclonal , 1:1000.

Techniques:

Journal: Cells

Article Title: Flaxseed Oil Alleviates Trimethyltin-Induced Cell Injury and Inhibits the Pro-Inflammatory Activation of Astrocytes in the Hippocampus of Female Rats

doi: 10.3390/cells13141184

Figure Lengend Snippet: FSO affects the expression of components of glutamatergic transmission 21 days post TMT intoxication. Relative protein abundances of GluN1 ( a ), GluN2A ( c ), GLT-1 ( d ), GluN2b ( e ), and PSD-95 ( f ) in the crude hippocampal membrane fractions of control (Ctrl), flaxseed oil (FSO)-treated, trimethyltin (TMT)-treated, and flaxseed oil-treated TMT-intoxicated (FSO+TMT) animals assessed by Western blot analysis. Bars represent mean protein abundance relative to β-actin ± SD, from n = 5 hippocampi per group. ( b ) Representative immunoblots of investigated proteins of Ctrl, FSO, TMT, and FSO+TMT animals. β-actin was probed to ensure equal protein loading. Significance shown inside the graphs: * p < 0.05 or less relative to Ctrl; # p < 0.05 or less relative to TMT.

Article Snippet: Anti- NMDA Receptor 2A (GluN2A) , Merck Millipore (Burlington, Massachusetts, USA), rabbit monoclonal , 1:1000.

Techniques: Expressing, Transmission Assay, Membrane, Control, Western Blot, Quantitative Proteomics