Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Activation of ERK by anandamide in dormant blastocysts via CB1. (A) Localization of CB1 in dormant and activated blastocysts. The trophectoderm cell surface is decorated with CB1. The levels of CB1 are significantly lower in activated blastocysts than those in dormancy. (B) Rapid activation of ERK by anandamide (ANA) in blastocysts. Dormant blastocysts were cultured in vitro in the presence of 7 nM anandamide for the indicated times in minutes. Increased phosphorylation of ERK1/2(p-ERK1/2) and its translocation into nuclei were observed in dormant blastocyst trophectoderm cells within 5 min of their exposure to 7 nM anandamide, reaching a peak between 15 and 30 min. (C) Activation of ERK by anandamide is dose-dependent. Dormant blastocysts were cultured in vitro in the presence of 7 or 28 nM anandamide for 15 min. Anandamide at 7 nM activated ERK1/2 in dormant blastocyst trophectoderm cells, whereas it failed to do so at 28 nM. A CB1-selective antagonist SR141716A (SR1) at 7 nM or a MEK1/2 inhibitor U0126 at 1 μM inhibited the activation of ERK1/2 by 7 nM anandamide. (D) Total ERK remained unchanged. No changes in immunointensity for total ERK1/2 were observed in dormant blastocysts exposed to 7 nM anandamide or the vehicle. (E–G) Differential activation of ERK signaling by anandamide in CB mutant dormant blastocysts. CB1–/–, CB2–/–,or CB1–/– × CB2–/– dormant blastocysts were cultured in vitro in the presence of 7 nM anandamide. Activation of ERK1/2 in the presence of 7 nM anandamide for 15 min was abrogated by the CB1 antagonist SR141716A (SR1) in CB2–/– blastocyst, but not in CB1–/– or CB1–/– × CB2–/– blastocysts. Images shown depict TRITC-labeled antigens in red, Hoechst-labeled nuclei in blue, and the merge in pink. (Scale bars, 20 μm.)

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Activation Assay, Cell Culture, In Vitro, Translocation Assay, Mutagenesis, Labeling

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Activation of ERK by anandamide in normal day-4 blastocysts via CB1. Day-4 blastocysts were cultured in vitro in the presence of 7 or 28 nM anandamide (ANA) for 15 min. Activation of ERK1/2 at lower (7 nM) but not higher (28 nM) anandamide concentration was observed. A CB1-selective antagonist SR141716A (SR1) inhibited the accumulation of phospho-ERK1/2 (p-ERK1/2) by 7 nM anandamide. Images depict TRITC-labeled antigen in red, Hoechst-labeled nuclei in blue, and the merge in pink. (Scale bar, 20 μm.) Tr, trophectoderm; ICM, inner cell mass.

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Activation Assay, Cell Culture, In Vitro, Concentration Assay, Labeling

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Cannabinoid agonist CP55,940 induces activation of ERK in differentiating TS cells via CB1. (A) CB1 is expressed in TS cells. This cell line is stably transfected with the GFP gene. Images depict GFP in green, CB1 in red (TS cell surfaces), and the merge in yellow. (Scale bar, 50 μm.) (B and C) Activation of ERK in TS cells by CP55,940 (CP). TS cells were plated and expanded for 48 h. The cells were serum-starved for 5 h then exposed to different concentrations of CP for 15 min or 7 nM CP for the indicated times or to CP at 7 nM in the presence or absence of a MEK1/2 inhibitor (U0126), CB1-selective antagonist SR141716A (SR1), or CB2-selective antagonist SR144528 (SR2) for 5 min. Phosphorylation of ERK1 in differentiating TS cells was rapidly induced by 7 nM CP. U0126 or SR1, but not SR2, inhibited this activation. Quantitative analysis of ERK activation in C is expressed as percentage relative to the maximum band intensity.

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Activation Assay, Stable Transfection, Transfection

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Inhibition of depolarization-induced Ca2+ influx by 28 nM anandamide in dormant blastocysts. (A) Distribution of Ca2+ channel α subunits, α1B (N-type) and α1C (L-type), in dormant blastocysts. Both inner cell mass (ICM) and trophectoderm cell (Tr) are decorated with α1B and α1C subunits shown in red, Hoechst-labeled nuclei in blue, and the merge in pink. (B) Inhibition of depolarization-induced Ca2+ influx by anandamide (ANA). Ca2+ mobilization in blastocysts was visualized with Fluo-4 acetoxymethyl ester. Depolarization-induced Ca2+ influx in dormant blastocysts after exposure to 60 mM KCl was dramatically inhibited by 28 nM anandamide but not by 7 nM. The CB1-selective antagonist SR141716A (SR1) at equimolar concentration reversed this inhibition, but the CB2-selective antagonist SR144528 (SR2) was ineffective. The relative level of intracellular Ca2+ is indicated by the fluorescent intensity, which is displayed in pseudocolor according to the color bar by using lsmib. (Scale bar, 20 μm.)

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Inhibition, Labeling, Concentration Assay

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Anandamide at 7 nM confers blastocyst competency to implantation via CB1

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

doi: 10.1073/pnas.1310145110

Figure Lengend Snippet: The D1R–NMDAR interaction bidirectionally regulates the surface distribution and dynamics of D1R and NMDAR. (A) Immunostaining of surface D1R-CFP (green) and GluN1 subunit (red) in hippocampal neurons. The yellow arrow shows overlay. (B) Immunostaining of surface D1R-CFP in control or after D1/5R agonist, TAT-t2, or TAT-[N2A15] application. (Scale bar, 250 nm.) (C) Normalized measures of D1R-CFP clusters intensity in control (n = 32 neuronal fields), D1/5R agonist-treated (n = 24 neuronal fields; *P < 0.05 compared with control), TAT-NSt2–treated (non-sense of TAT-t2, n = 19 neuronal fields), TAT-t2–treated (n = 21 neuronal fields; **P < 0.01 compared with TAT-NSt2), TAT-NSt3–treated (non-sense of TAT-t3, n = 11 neuronal fields), TAT-t3–treated (n = 12 neuronal fields; P > 0.05 compared with TAT-NSt3), TAT-[NS15]–treated (n = 27 neuronal fields; P > 0.05), or TAT-[N2A15]–treated (n = 21 neuronal fields, *P < 0.05 compared with TAT-[NS15]) conditions. (D) Representative trajectories (1,000 frames, 20-Hz acquisition rate) of surface single D1R-CFP (Left) (green) (scale bar, 400 nm) and GluN1-NMDAR (Right) (blue) (scale bar, 300 nm) in the absence and presence of either D1/5R agonist (10 µM, 15 min) or TAT-t2 (10 µM, 15 min). Bold dotted line, perisynaptic area; thin dotted line, PSD area. (E) Plot of the MSD of surface D1R-CFP (Upper) (green) and GluN1-NMDAR (Lower) (blue) versus time in presence of TAT-NS or TAT-t2 peptides (10 µM, 15 min). The SEM is included for each data point (D1R: TAT-NS, n = 986 trajectories, and TAT-t2, n = 1,326; GluN1-NMDAR: TAT-NS, n = 198, and TAT-t2, n = 134). (F and G) Representative surface distributions of single D1R-CFP (green) (F) and GluN1-NMDAR (blue) (G) in the synaptic area (PSD + perisynaptic area) in control, D1/5R agonist, and TAT-t2 conditions. Each dot represents the detection of a single receptor during a frame. Comparisons of the time spent in the synaptic area (dwell time) by single D1R-CFP (control, n = 173 trajectories; D1/5R agonist, n = 142, **P < 0.01; TAT-t2, n = 752, *P < 0.05) (F) and GluN1-NMDAR (control, n = 189 trajectories; D1/5R agonist, n = 157, *P < 0.05; TAT-t2, n = 134, **P < 0.01) (G) and the synaptic fraction of detected single D1R-CFP (control, n = 14 neuronal fields; D1/5R agonist, n = 19, **P < 0.01; D1/5R agonist in the presence of dynasore, n = 47, **P < 0.01; TAT-t2, n = 15, ***P < 0.001) (F), D5R-CFP (n = 16, P > 0.05) (F), and GluN1-NMDAR (control, n = 11; D1/5R agonist, n = 15, *P < 0.05; TAT-t2, n = 14, *P < 0.05) (G). Dyn., dynasore; D1/5 ago., D1/5 receptor agonist SKF-38393.

Article Snippet: For single-nanoparticle tracking, QD 655 coupled to goat anti-rabbit F(ab′) 2 or anti-mouse IgG (Invitrogen) was incubated (1:10,000, 10 min) onto neurons previously exposed for 10 min to either mouse monoclonal anti-GFP (1 µg; Invitrogen), rabbit polyclonal anti-D1R (1 µg; Lifespan Biosciences), mouse monoclonal anti-GluA2:00 AMPAR subunit (1 µg; Millipore), or rabbit polyclonal anti–GluN1-NMDAR subunit (1 µg; Alomone Laboratories) antibodies.

Techniques: Immunostaining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

doi: 10.1073/pnas.1310145110

Figure Lengend Snippet: D1R activation or D1R/GluN1-NMDAR interaction blockade increases synaptic NMDAR content and favors AMPAR synaptic long-term potentiation. (A) (Left) Excitatory postsynaptic current traces recorded at −70 mV and +40 mV from a representative hippocampal CA1 pyramidal cell, before and 10 min after exposure to D1/5R agonist. (Right) Relative change over time of the AMPA/NMDA ratio at CA1 synapses in the absence or presence of D1/5R agonist (n = 13, *P < 0.05 10 min after agonist) and in the absence or presence of vehicle (n = 7, P > 0.05). (B) Surface imaging of GluN1-SEP in neurons incubated with either TAT-NS or TAT-t2 (10 µM). (Scale bar, 5 µm.) (Right) Average value of GluN1-SEP content in the synaptic area after TAT-NS or TAT-t2 application (n = 8 neurons per group, **P < 0.01). (C) Dendritic fragment of a hippocampal neuron expressing Homer 1c-DsRed (Upper) and GluA1-SEP (Lower). SEP only fluoresces at neutral pH when receptors are inserted at the plasma membrane. Ten minutes after chemical LTP induction (cLTP), the GluA1-SEP fluorescence intensity increased in postsynaptic clusters. (Insets) High magnification of a synaptic GluA1-SEP cluster. (Scale bar, 2 µm.) (D) Comparison of the synaptic GluA1-SEP fluorescence intensity before and after cLTP with prior TAT-NS (n = 198 synapses, *P < 0.05) or TAT-t2 (n = 215 synapses, *P < 0.05) (TAT-NS versus TAT-t2; *P < 0.05) application. (E) Schematic model of the D1R–NMDAR surface interplay in hippocampal neurons. D1Rs are highly diffusive at the neuronal surface and are dynamically retained in clusters in the vicinity of glutamate synapses where they interact with NMDAR. Dopamine release disrupts this interaction and favors the lateral redistribution of both receptors: D1Rs freely explore extrasynaptic areas, whereas NMDARs laterally reach the PSD where they impact on the long-term plasticity of glutamate synapses.

Article Snippet: For single-nanoparticle tracking, QD 655 coupled to goat anti-rabbit F(ab′) 2 or anti-mouse IgG (Invitrogen) was incubated (1:10,000, 10 min) onto neurons previously exposed for 10 min to either mouse monoclonal anti-GFP (1 µg; Invitrogen), rabbit polyclonal anti-D1R (1 µg; Lifespan Biosciences), mouse monoclonal anti-GluA2:00 AMPAR subunit (1 µg; Millipore), or rabbit polyclonal anti–GluN1-NMDAR subunit (1 µg; Alomone Laboratories) antibodies.

Techniques: Activation Assay, Imaging, Incubation, Expressing, Fluorescence

Journal: Molecular Imaging

Article Title: PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection

doi: 10.1155/2021/9982020

Figure Lengend Snippet: MicroPET imaging of S1PR1 activity in S aureus -infected mice. (a) Radiosynthesis of S1PR1-specific radiotracer, [ 18 F]TZ4877; (b) representative sagittal microPET images of [ 18 F]TZ4877 in mice. Comparing with sham mice, the tracer uptake was significantly higher in the infected mice, and the increased uptake of the tracer showed S aureus dose dependent; (c) the tracer uptake in the brain was quantified; time-activity curves showed that the tracer uptake in infected mice was significantly higher than mice without infections; (d) the average tracer uptake in the brain from 30 to 50 min of the PET scan showed a dose-dependent manner. Data represent the mean ± SEM, n = 3 for each group.

Article Snippet: After washing in PBS, all sections were then incubated with rabbit anti-S1PR1 (Alomone, Israel) antibody overnight at 4°C and then washed and incubated with ImmPRESS HRP Horse anti-rabbit polymer for 1 hour at room temperature and developed with ImmPACT DAB (Vector Laboratories, Burlingame, CA).

Techniques: Imaging, Activity Assay, Infection

Journal: Molecular Imaging

Article Title: PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection

doi: 10.1155/2021/9982020

Figure Lengend Snippet: Biodistribution (%ID/g, mean ± SEM) of S1PR1-specific [ 18 F]TZ4877 in Balb/c mice ( n = 4).

Article Snippet: After washing in PBS, all sections were then incubated with rabbit anti-S1PR1 (Alomone, Israel) antibody overnight at 4°C and then washed and incubated with ImmPRESS HRP Horse anti-rabbit polymer for 1 hour at room temperature and developed with ImmPACT DAB (Vector Laboratories, Burlingame, CA).

Techniques: Mouse Assay

Journal: Molecular Imaging

Article Title: PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection

doi: 10.1155/2021/9982020

Figure Lengend Snippet: Biodistribution of S1PR1-specific [ 18 F]TZ4877 in sham, infected, and infected with treatments mice ( n = 4).

Article Snippet: After washing in PBS, all sections were then incubated with rabbit anti-S1PR1 (Alomone, Israel) antibody overnight at 4°C and then washed and incubated with ImmPRESS HRP Horse anti-rabbit polymer for 1 hour at room temperature and developed with ImmPACT DAB (Vector Laboratories, Burlingame, CA).

Techniques: Infection, Mouse Assay

Journal: Molecular Imaging

Article Title: PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection

doi: 10.1155/2021/9982020

Figure Lengend Snippet: MicroPET imaging of S1PR1 activity in S aureus -infected mice. (a) Representative sagittal microPET images of [ 18 F]TZ4877 in the hind limb of mice. The tracer uptake was relatively low in the hind limb muscle with a SUV of ~1.5 in sham mice. Comparing with sham mice, the tracer uptake was significantly higher in the hind limb of infected mice; (b) time-activity curves showed that the tracer uptake in infected mice was significantly higher than sham mice; (c) the average tracer uptake in the hind limb muscle from 30 to 50 min of the PET scan showed a ~39% increase of SUV in infected mice with a P value of 0.0082. Data represent the mean ± SEM, n = 3 for each group.

Article Snippet: After washing in PBS, all sections were then incubated with rabbit anti-S1PR1 (Alomone, Israel) antibody overnight at 4°C and then washed and incubated with ImmPRESS HRP Horse anti-rabbit polymer for 1 hour at room temperature and developed with ImmPACT DAB (Vector Laboratories, Burlingame, CA).

Techniques: Imaging, Activity Assay, Infection

Journal: Molecular Imaging

Article Title: PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection

doi: 10.1155/2021/9982020

Figure Lengend Snippet: PET measurements of S1PR1-specific [ 18 F]TZ4877 in S aureus -infected and sham mice.

Article Snippet: After washing in PBS, all sections were then incubated with rabbit anti-S1PR1 (Alomone, Israel) antibody overnight at 4°C and then washed and incubated with ImmPRESS HRP Horse anti-rabbit polymer for 1 hour at room temperature and developed with ImmPACT DAB (Vector Laboratories, Burlingame, CA).

Techniques: Infection

Journal: Molecular Imaging

Article Title: PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection

doi: 10.1155/2021/9982020

Figure Lengend Snippet: Immunohistochemistry analysis of S1PR1 in hind limb muscle of sham and S aureus -infected mice. S1PR1 was significantly upregulated in the muscle of infected mice (red arrow) comparing with sham mice (green arrow), scale bar = 100 μ m.

Article Snippet: After washing in PBS, all sections were then incubated with rabbit anti-S1PR1 (Alomone, Israel) antibody overnight at 4°C and then washed and incubated with ImmPRESS HRP Horse anti-rabbit polymer for 1 hour at room temperature and developed with ImmPACT DAB (Vector Laboratories, Burlingame, CA).

Techniques: Immunohistochemistry, Infection

Journal: The Journal of Neuroscience

Article Title: BRAG2a Mediates mGluR-Dependent AMPA Receptor Internalization at Excitatory Postsynapses through the Interaction with PSD-95 and Endophilin 3

doi: 10.1523/JNEUROSCI.1645-19.2020

Figure Lengend Snippet: Antibodies used in the present study

Article Snippet: Two days after the transfection, neurons were treated with the conditioned medium containing 0.1 μ m TTX for 30 min, followed by incubation with 50 μ m DHPG for 15 min and the conditioned medium for 45 min. To label surface AMPA and NMDA receptors, cultured neurons were incubated with either GluA1 (extracellular) antibody (1.0 μg/ml; Alomone Labs) or GluN2B (extracellular) antibody (1.0 μg/ml; Watanabe et al., 1998 ) at 10°C for 30 min, washed quickly with warmed neuronal maintenance medium, and then subjected to immunostaining.

Techniques: Recombinant, Immunohistochemistry-IF, Transduction

Journal: The Journal of Neuroscience

Article Title: BRAG2a Mediates mGluR-Dependent AMPA Receptor Internalization at Excitatory Postsynapses through the Interaction with PSD-95 and Endophilin 3

doi: 10.1523/JNEUROSCI.1645-19.2020

Figure Lengend Snippet: The interaction of BRAG2a with endophilin 3 and PSD-95 is important for the mGluR-dependent decrease in AMPAR surface level. A, B, The effect of BRAG2 knockdown on basal surface GluA1 level. A, Representative immunofluorescence images of dendritic shafts and spines of cultured hippocampal neurons transfected with shBRAG2 (BRAG2-KD) or shControl (control) vectors at DIV14 and subjected to surface AMPAR labeling with anti-GluA1 (extracellular) antibody at DIV16. B, Quantification of surface GluA1 levels. Immunofluorescence intensity of surface GluA1 in BRAG2-KD-transfected neurons was normalized to that in the control-transfected neurons. Note that knockdown of BRAG2 did not affect the basal surface GluA1 levels. C, D, The effect of BRAG2 knockdown on surface GluA1 levels following DHPG treatment. D, Quantification of surface GluA1 levels. Note that BRAG2 knockdown blocked the decrease in surface GluA1 levels following 50 μm DHPG treatment (DHPG 50). Asterisks indicate statistical significance (one-way ANOVA; F(2,61) = 4.507, p = 0.0149; followed by Tukey–Kramer post hoc test, *p < 0.05). E, F, The effect of BRAG2 knockdown on surface GluN2B levels following DHPG treatment. E, Representative immunofluorescence images of dendritic shafts and spines of BRAG2-KD or control hippocampal neurons treated with 50 μm DHPG and subjected to surface NMDAR labeling with anti-GluN2B (extracellular) antibody. F, Quantification of surface GluN2B levels; the immunofluorescence intensities of surface GluN2B in BRAG2-KD and control neurons treated with DHPG were normalized to that in control and DHPG-untreated (DHPG 0) neurons. Note that DHPG treatment did not change surface GluN2B levels in BRAG2-KD or control neurons (one-way ANOVA: F(2,59) = 0.964, p = 0.387). Data for each surface GluN2B were obtained from 20–22 transfected neurons from three plates, and these results were confirmed by three independent experiments. G, H, The effect of coexpression of shBRAG2 and BRAG2a or its mutants on the DHPG-induced decrease in surface GluA1 levels. G, Representative immunofluorescence images of dendritic shafts and spines of neurons transfected with BRAG2-KD alone or with BRAG2-KD and sh-res BRAG2a-WT, BRAG2b-WT, BRAG2a-P956/957A, BRAG2a-ΔSTVV, or BRAG2a-ΔSec7. H, Quantification of surface GluA1 levels. Note that the BRAG2-knock-down phenotype on DHPG-induced surface GluA1 levels could be rescued by coexpression of shRNA-resistant BRAG2a-WT, but not BRAG2b-WT, BRAG2a-P956/957A, BRAG2a-ΔSTVV, or BRAG2a-ΔSec7. Asterisks indicate statistical significance (one-way ANOVA: F(5,138) = 12.822, p < 0.0001; followed by Tukey–Kramer post hoc test, *p < 0.05). Each surface GluA1 data were obtained from three independent experiments, and 20–28 transfected neurons from three plates were analyzed in each experiment. Scale bars, 2 μm.

Article Snippet: Two days after the transfection, neurons were treated with the conditioned medium containing 0.1 μ m TTX for 30 min, followed by incubation with 50 μ m DHPG for 15 min and the conditioned medium for 45 min. To label surface AMPA and NMDA receptors, cultured neurons were incubated with either GluA1 (extracellular) antibody (1.0 μg/ml; Alomone Labs) or GluN2B (extracellular) antibody (1.0 μg/ml; Watanabe et al., 1998 ) at 10°C for 30 min, washed quickly with warmed neuronal maintenance medium, and then subjected to immunostaining.

Techniques: Immunofluorescence, Cell Culture, Transfection, Labeling, shRNA

Journal: The Journal of Neuroscience

Article Title: BRAG2a Mediates mGluR-Dependent AMPA Receptor Internalization at Excitatory Postsynapses through the Interaction with PSD-95 and Endophilin 3

doi: 10.1523/JNEUROSCI.1645-19.2020

Figure Lengend Snippet: Lateral distribution of BRAG2a, PSD-95, GluA1/2, Arf6, endophilin 3, AP-2, and clathrin along the postsynaptic membrane. A, B, D, E, G, H, J, K, M, N, P, Q, S, and T, Representative postembedding immunoelectron microscopic images of the distribution of BRAG2a (A, B), PSD-95 (D, E), GluA1/2 (G, H), Arf6 (J, K), endophilin 3 (M, N), AP-2/α-adaptin (P, Q), and clathrin (S, T) in excitatory asymmetric synapses of the mouse hippocampal CA1 region. C, F, I, L, O, R, and U, Histograms showing lateral distribution of immunogold particles for BRAG2a (C), PSD-95 (F), GluA1/2 (I), Arf6 (L), endophilin 3 (O), AP-2/α-adaptin (R), and clathrin (U) along the postsynaptic membrane. The edge of the postsynaptic density (arrows) is defined as 0. The bin size of the histogram is 25 nm, and the synaptic and extrasynaptic sites are shown in the left and right sides, respectively. Scale bars, 100 nm.

Article Snippet: Two days after the transfection, neurons were treated with the conditioned medium containing 0.1 μ m TTX for 30 min, followed by incubation with 50 μ m DHPG for 15 min and the conditioned medium for 45 min. To label surface AMPA and NMDA receptors, cultured neurons were incubated with either GluA1 (extracellular) antibody (1.0 μg/ml; Alomone Labs) or GluN2B (extracellular) antibody (1.0 μg/ml; Watanabe et al., 1998 ) at 10°C for 30 min, washed quickly with warmed neuronal maintenance medium, and then subjected to immunostaining.

Techniques:

Journal: The Journal of Neuroscience

Article Title: BRAG2a Mediates mGluR-Dependent AMPA Receptor Internalization at Excitatory Postsynapses through the Interaction with PSD-95 and Endophilin 3

doi: 10.1523/JNEUROSCI.1645-19.2020

Figure Lengend Snippet: The effect of DHPG treatment on BRAG2a, PSD-95, and GluA1/2 levels in the PSD and extrasynaptic region using acute hippocampal slice cultures. A, B, The effect of the DHPG treatment on the expression of GluA1/2 and Arc/Arg3.1, and the phosphorylation of p38 MAPK in acute hippocampal slice cultures. A, Representative immunoblots of acute hippocampal slices treated with or without DHPG with anti-GluA1/2, anti-Arc/Arg3.1, anti-phospho-p38 MAPK, anti-p38 MAPK, and anti-α-tubulin antibodies. B, Quantification of immunoreactive intensities of GluA1/2, Arc/Arg3.1, and phospho/total p38 MAPKs. Each immunoreactive intensity was normalized by the respective intensity for α-tubulin and expressed as the ratio with the control value. Note that the DHPG treatment induced the significant upregulation of Arc/Arg3.1 and the phosphorylation of p38 MAPK in hippocampal slices. Data for each group were obtained from three culture plates (n = 3). These results were confirmed by three independent experiments. C–T, Representative postembedding immunoelectron microscopic images (C–F, I–L, O–R) and histograms (G, H, M, N, S, T) of the lateral distribution of immunogold particles for BRAG2a (C–H), PSD-95 (I–N), and GluA1/2 (O–T) in axospinous asymmetric synapses in the CA1 stratum radiatum of acute mouse hippocampal slices following 50 μm DHPG treatment. The edge of the postsynaptic density (arrowheads) is defined as 0. The bin size of the histogram is 25 nm. U, V, Quantification of immunogold particles for BRAG2a, PSD-95, and GluA1/2 in the PSD (U) and extrasynaptic (V) region along the postsynaptic membrane. Values in U and V are expressed as the average immunogold particle numbers in the PSD of axospinous synapses and the percentage of immunogold particles in the extrasynaptic region in total immunoreactive particles along the postsynaptic membrane, respectively. Note that the DHPG treatment decreased the immunogold particles for GluA1/2 but not BRAG2a or PSD-95 without any changes in the proportion of BRAG2a, PSD-95, or GluA1/2 in the extrasynaptic region. *p < 0.05 (t test). Data for each group were obtained from three slices (n = 3). These results were confirmed by three independent experiments from different mice. Scale bars, 100 nm.

Article Snippet: Two days after the transfection, neurons were treated with the conditioned medium containing 0.1 μ m TTX for 30 min, followed by incubation with 50 μ m DHPG for 15 min and the conditioned medium for 45 min. To label surface AMPA and NMDA receptors, cultured neurons were incubated with either GluA1 (extracellular) antibody (1.0 μg/ml; Alomone Labs) or GluN2B (extracellular) antibody (1.0 μg/ml; Watanabe et al., 1998 ) at 10°C for 30 min, washed quickly with warmed neuronal maintenance medium, and then subjected to immunostaining.

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: CX 3 CR1 Is Expressed by Human B Lymphocytes and Meditates CX 3 CL1 Driven Chemotaxis of Tonsil Centrocytes

doi: 10.1371/journal.pone.0008485

Figure Lengend Snippet: ( A ) Flow cytometric analysis of CX 3 CR1 expression in tonsil and blood B cells. Representative histograms from each B cell fractions, and THP-1 and Raji cell lines, tested as positive and negative controls, respectively, are shown. ( B ) Quantization of CX 3 CR1 by real time PCR in peripheral blood and tonsil B cells. Data are normalized to the expression of POLR2A. Values are expressed as arbitrary units calculated as fold of CX 3 CR1 expression relative to the THP-1 cell line, arbitrarily set at 1. Raji cell line was tested as negative control. Data are median, minimum and maximum values from two different experiments performed in quadruplicate. ( C ) Displacement experiments of 125 I-CX 3 CL1 in tonsil and blood B cells. Left panel . Cells were incubated for 2 h at 4°C with 1 nM 125 I-CX 3 CL1 in the absence or presence of 1, 10 and 100 nM cold CX 3 CL1. Percentage of binding inhibition by unlabeled CX 3 CL1, calculated as ratio between cell-bound cpm in the presence of unlabeled ligand and cell-bound cpm in the absence of unlabeled ligand multiplied by 100, was used as a measure for competition between 125 I-labeled and unlabelled CX 3 CL1. THP-1 and Raji cell lines were tested as positive and negative controls, respectively. Right panel . The experiments shown in the left panel were repeated using 100 nM cold CXCL8 as negative control.

Article Snippet: CD10-FITC (MEM78 clone), anti-human IgD-FITC, CD95-FITC, CD44-FITC, anti-Bcl-2-FITC, and anti-Ki-67-FITC mAbs from DAKO (Glostrup, Denmark); unconjugated CD77 and CD39 mAbs from Immunotech, Marseille, France; unconjugated rabbit anti-human CX 3 CR1 (Serotec) that detects an epitope starting from 175 to 189 amino acids; unconjugated CD3, CD56, CD68, CD10, CD27, and anti-human IgM mAbs from DAKO.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Negative Control, Incubation, Binding Assay, Inhibition, Labeling

Journal: PLoS ONE

Article Title: CX 3 CR1 Is Expressed by Human B Lymphocytes and Meditates CX 3 CL1 Driven Chemotaxis of Tonsil Centrocytes

doi: 10.1371/journal.pone.0008485

Figure Lengend Snippet: ( A ) Apoptosis evaluation in purified tonsil GC B cells. The proportion of early apoptotic GC B cells was detected by Annexin V staining at time 0 and after 1, 2, 3, and 4h culture. Results are expressed as median, minimum and maximum values from five different GC B cell suspensions. ( B ) Flow cytometric analysis of CX 3 CR1 expression on freshly purified tonsil GC, naïve, and memory B cells. Results are expressed in box plot as median percent positive cells, minimum and maximum values, and quartiles, from ten different experiments. ( C ) Chemotaxis of GC, naïve, and memory B lymphocytes to rCX 3 CL1. Results are median numbers of migrated cells, maximum and minimum values, from five different experiments for each B cell subset. * P = 0.043 for both 300 and 600 ng/ml rCX 3 CL1. ▪ = Chemotaxis of non-GC B cells to 300 ng/ml rCXCL12 tested as control. ( D ) Freshly isolated GC B cells were pre-incubated with or without PTX and subjected to chemotaxis to 300 ng/ml CX 3 CL1 or medium (nil). Results are median numbers of migrated cells, minimum and maximum values from three different experiments.

Article Snippet: CD10-FITC (MEM78 clone), anti-human IgD-FITC, CD95-FITC, CD44-FITC, anti-Bcl-2-FITC, and anti-Ki-67-FITC mAbs from DAKO (Glostrup, Denmark); unconjugated CD77 and CD39 mAbs from Immunotech, Marseille, France; unconjugated rabbit anti-human CX 3 CR1 (Serotec) that detects an epitope starting from 175 to 189 amino acids; unconjugated CD3, CD56, CD68, CD10, CD27, and anti-human IgM mAbs from DAKO.

Techniques: Purification, Staining, Expressing, Chemotaxis Assay, Control, Isolation, Incubation

Journal: PLoS ONE

Article Title: CX 3 CR1 Is Expressed by Human B Lymphocytes and Meditates CX 3 CL1 Driven Chemotaxis of Tonsil Centrocytes

doi: 10.1371/journal.pone.0008485

Figure Lengend Snippet: ( A ) CX 3 CR1 + and CX 3 CR1 − GC B cells were analyzed after staining with mAbs. Results are percent positive cells, minimum to maximum ranges from ten independent experiments. ( B ) V H 5(D)J H -μ rearrangement sequences were evaluated in CX 3 CR1 + and CX 3 CR1 − GC B cells. 104 molecular clones from five different CX 3 CR1 + GC B cell fractions were compared to 63 molecular clones from three different CX 3 CR1 − GC B lymphocyte fractions. 34 molecular clones from three different CD10 - CD27 − naive B cell fractions were tested as controls. Results are ratios between number of mutations and number of molecular clones.

Article Snippet: CD10-FITC (MEM78 clone), anti-human IgD-FITC, CD95-FITC, CD44-FITC, anti-Bcl-2-FITC, and anti-Ki-67-FITC mAbs from DAKO (Glostrup, Denmark); unconjugated CD77 and CD39 mAbs from Immunotech, Marseille, France; unconjugated rabbit anti-human CX 3 CR1 (Serotec) that detects an epitope starting from 175 to 189 amino acids; unconjugated CD3, CD56, CD68, CD10, CD27, and anti-human IgM mAbs from DAKO.

Techniques: Staining, Clone Assay

Journal: PLoS ONE

Article Title: CX 3 CR1 Is Expressed by Human B Lymphocytes and Meditates CX 3 CL1 Driven Chemotaxis of Tonsil Centrocytes

doi: 10.1371/journal.pone.0008485

Figure Lengend Snippet: ( A ) Double staining of CX 3 CR1 + GC B cells with CD27 and CD23 mAbs. Each B cell fraction was further characterized with mAbs (CD27 + cells, right lower panel; CD23 + cells, left lower panel). Results are median percent positive cells, maximum and minimum values from ten different experiments. ( B ) CX 3 CR1 + GC B cells were subjected to chemotaxis to 300 ng/ml rCX 3 CL1. Migrated cells were double stained for CD27 and CD23. One representative experiment out of seven is shown. ( C ) Purified GC B cells were subjected to CX 3 CL1-driven chemotaxis and migrated cells were collected and stained with a panel of mAbs. Results are median percent positive cells, maximum and minimum values from four different experiments.

Article Snippet: CD10-FITC (MEM78 clone), anti-human IgD-FITC, CD95-FITC, CD44-FITC, anti-Bcl-2-FITC, and anti-Ki-67-FITC mAbs from DAKO (Glostrup, Denmark); unconjugated CD77 and CD39 mAbs from Immunotech, Marseille, France; unconjugated rabbit anti-human CX 3 CR1 (Serotec) that detects an epitope starting from 175 to 189 amino acids; unconjugated CD3, CD56, CD68, CD10, CD27, and anti-human IgM mAbs from DAKO.

Techniques: Double Staining, Chemotaxis Assay, Staining, Purification

Journal: PLoS ONE

Article Title: CX 3 CR1 Is Expressed by Human B Lymphocytes and Meditates CX 3 CL1 Driven Chemotaxis of Tonsil Centrocytes

doi: 10.1371/journal.pone.0008485

Figure Lengend Snippet: ( A ) Splenocytes from WT mice double stained with anti-CX 3 CR1 and B220 mAbs were analyzed by flow cytometry. Histograms from two different WT mice are shown. ( B ) Splenocytes from WT mice were tested for chemotaxis to murine rCX 3 CL1 or rCXCL12 (control). Migrated B cells were enumerated by flow cytometry using anti-B220 mAb. Results are median number of migrated cells, minimum and maximum values from four different experiments. ( C ) CX 3 CR1 − / − , CX 3 CL1 − / − or WT mice were immunized with OVA and tested at different dilutions for specific IgG production by ELISA. Results were expressed as median optical densities (OD), maximum and minimum values, from ten different experiments. Serum from two WT mice was titrated for OVA IgG antibodies (1: 200, 1:500, 1:1000, 1:10000, 1:50000, 1:100000 dilutions) and used in each tests as standard curve, whereas serum from mice before immunization was used as negative control.

Article Snippet: CD10-FITC (MEM78 clone), anti-human IgD-FITC, CD95-FITC, CD44-FITC, anti-Bcl-2-FITC, and anti-Ki-67-FITC mAbs from DAKO (Glostrup, Denmark); unconjugated CD77 and CD39 mAbs from Immunotech, Marseille, France; unconjugated rabbit anti-human CX 3 CR1 (Serotec) that detects an epitope starting from 175 to 189 amino acids; unconjugated CD3, CD56, CD68, CD10, CD27, and anti-human IgM mAbs from DAKO.

Techniques: Staining, Flow Cytometry, Chemotaxis Assay, Control, Enzyme-linked Immunosorbent Assay, Negative Control