Journal: International Journal of Molecular Sciences

Article Title: NR1 and NR3B Composed Intranuclear N -methyl- d -aspartate Receptor Complexes in Human Melanoma Cells

doi: 10.3390/ijms19071929

Figure Lengend Snippet: Western blot analysis gave detailed picture on NMDAR subunit protein expression. NR1-1a and NR2A subunits were expressed in melanocytes, while other subunits were undetectable by western blots ( A ). Fractionated melanoma samples revealed that NR1 and NR3B subunits are expressed in every cellular compartment ( B ). In each melanoma cell line NR2A and NR3A were present in the cytosol and membranes, but in the nucleus ( B ). NR2B was undetectable in each compartment ( B ). Immunoblot signals of NR1-1a were detected in the cytosol and membranes, but more importantly also in the nuclei of melanoma cells ( B ). Controls used in the experiments: MITF (microphthalmia associated transcription factor) for melanocyte differentiation control; GAPDH as an internal control for melanocyte whole cell lysates; actin as internal controls for the comparison of the cellular fractions; TATABP (TATA box binding protein) expression as internal control for nuclear fraction analysis. The positive control for detection of NMDAR subunit protein expression was human brain tissue lysate (see ). Whole membrane pictures of western blots are presented as . Normalization of the relative optical density parameters of the protein subunit expressions to the respective control is shown in a graphical format in the . (C = cytosolic, M = membrane, N = nuclear fractions of melanoma cells).

Article Snippet: After that samples were washed again three times in PBS, and cultures were incubated with rabbit polyclonal anti-NR3B antibody (Alomone Labs, Jerusalem, Israel), at a dilution of 1:50 in PBST, at 4 °C overnight.

Techniques: Western Blot, Expressing, Binding Assay, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: NR1 and NR3B Composed Intranuclear N -methyl- d -aspartate Receptor Complexes in Human Melanoma Cells

doi: 10.3390/ijms19071929

Figure Lengend Snippet: Immunocytochemistry demonstrating colocalization of NR1 and NR3B in the nuclei of melanoma cells, but not in the cell membrane in melanocytes. Confocal microscopic evaluation revealed that NR1 (red) was present in the cytosol of every melanoma cell line, while NR3B (green) showed weaker and less diffuse signals in the cytoplasm. Unambiguous plasma membrane immunofluorescent signals were not visible, but NR1 and NR3B colocalized inside the nuclei of melanoma cells. NR1 and NR3B colocalization was absent or undetected in the nuclei of NHEM. That 1-μm thick optical section was selected for presentation which specifically went through the majority of nuclei to show that all nuclei were immunopositive. One representative cell pointed by white arrow was selected for the inserts at each part of the panel. Scale bar: 20 μm. Positive controls for the secondary antibodies and special controls for the primary antibodies were also performed (see ).

Article Snippet: After that samples were washed again three times in PBS, and cultures were incubated with rabbit polyclonal anti-NR3B antibody (Alomone Labs, Jerusalem, Israel), at a dilution of 1:50 in PBST, at 4 °C overnight.

Techniques: Immunocytochemistry

Journal: International Journal of Molecular Sciences

Article Title: NR1 and NR3B Composed Intranuclear N -methyl- d -aspartate Receptor Complexes in Human Melanoma Cells

doi: 10.3390/ijms19071929

Figure Lengend Snippet: Immunocytochemistry demonstrating nuclear colocalization of NR1-1a and NR3B in melanoma cells. Confocal microscopic analysis demonstrated that NR1-1a (red) shows similar nuclear colocalization in melanoma cells with NR3B (green) as NR1. Nuclei of NHEM showed very weak signals of NR1-1a. NR1-1a was present in the cytoplasmic region of NHEM but unequivocal colocalization with NR3B was not detected at any cellular compartment. As an example, two extra arrows are used in the insert that shows M35/01 cells: the dashed arrow points at an area where the immunofluorescent signal of NR1-1a (red) colocalizes with DAPI (blue), resulting in purple tone whereas the spotted arrow points at an area where NR1-1a colocalizes with DAPI and NR3B (green), resulting in orange colour. Photomicrographs of 1 μm thick optical sections passing through the majority of nuclei in the area of the interest are shown. One representative cell pointed by white arrow with numerous (nuclear) colocalizing signals was selected on each picture and presented in the insert. Scale bar: 20 μm. Positive controls for the secondary antibodies and special controls for the primary antibodies were also performed (See ).

Article Snippet: After that samples were washed again three times in PBS, and cultures were incubated with rabbit polyclonal anti-NR3B antibody (Alomone Labs, Jerusalem, Israel), at a dilution of 1:50 in PBST, at 4 °C overnight.

Techniques: Immunocytochemistry

Journal: International Journal of Molecular Sciences

Article Title: NR1 and NR3B Composed Intranuclear N -methyl- d -aspartate Receptor Complexes in Human Melanoma Cells

doi: 10.3390/ijms19071929

Figure Lengend Snippet: Nucleotide sequences, amplification sites, GenBank accession numbers, amplimer sizes and PCR reaction conditions for each primer pair are shown.

Article Snippet: After that samples were washed again three times in PBS, and cultures were incubated with rabbit polyclonal anti-NR3B antibody (Alomone Labs, Jerusalem, Israel), at a dilution of 1:50 in PBST, at 4 °C overnight.

Techniques: Amplification, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: NR1 and NR3B Composed Intranuclear N -methyl- d -aspartate Receptor Complexes in Human Melanoma Cells

doi: 10.3390/ijms19071929

Figure Lengend Snippet: Tables of antibodies used in the experiments.

Article Snippet: After that samples were washed again three times in PBS, and cultures were incubated with rabbit polyclonal anti-NR3B antibody (Alomone Labs, Jerusalem, Israel), at a dilution of 1:50 in PBST, at 4 °C overnight.

Techniques: Western Blot, Immunocytochemistry

Journal: Antioxidants

Article Title: Erythroid Differentiation Regulator 1 as a Regulator of Neuronal GSH Synthesis

doi: 10.3390/antiox13070771

Figure Lengend Snippet: Increased EAAC1 gene and protein expression in Neuro2a cells with Erdr1 siRNA knockdown. ( a ) Confocal images showing the effect of transfection with a specific siRNA targeting Erdr1 (siRNA) on the intensity of EAAC1 expression in Neuro2a cells compared with that in the negative control cells (scrambled siRNA). Scale bar, 100 μm. Insets in the panels are enlarged images corresponding to the box highlighted on the full images. Scale bar, 20 μm. ( b ) Quantification of relative EAAC1 densities in Neuro2a cells is shown. n = 5; 5–6 areas were measured in each sample. ** p < 0.01. ( c ) Protein expression of EAAC1 in Neuro2a cells transfected with a specific siRNA targeting Erdr1 (siRNA) and in the negative control cells (scrambled siRNA). Quantification of the EAAC1 protein expression (data in the left panel) by densitometry is shown in the right panel. n = 6. * p < 0.05.

Article Snippet: Non-specific staining was blocked with the reagent PBS containing 5% BSA, and the cells were incubated with anti-EAAC1 (Alomone Labs) at 1:1000 dilution for 1 h. After a wash with PBS-Tween20, the cells were labeled with fluorescent-labeled secondary antibodies Alexa-Fluor 488 anti-rabbit IgG (Invitrogen) at 1:1000 dilutions for 30 min.

Techniques: Expressing, Knockdown, Transfection, Negative Control

Journal: Antioxidants

Article Title: Erythroid Differentiation Regulator 1 as a Regulator of Neuronal GSH Synthesis

doi: 10.3390/antiox13070771

Figure Lengend Snippet: The schematic illustration indicates the relationship between Erdr1, GTRAP3-18, EAAC1, and GSH in the brain/hippocampus and Neuro2a/primary hippocampal neurons. In the brain/hippocampus of GTRAP3-18-deficient mice, GTRAP3-18 may be regulated by Erdr1 and exert compensatory feedback on Erdr1 expression. In Neuro2a/primary hippocampal neurons, knockdown of Erdr1 resulted in a decrease in GTRAP3-18, upregulation of EAAC1 expression, leading to an increase in GSH levels.

Article Snippet: Non-specific staining was blocked with the reagent PBS containing 5% BSA, and the cells were incubated with anti-EAAC1 (Alomone Labs) at 1:1000 dilution for 1 h. After a wash with PBS-Tween20, the cells were labeled with fluorescent-labeled secondary antibodies Alexa-Fluor 488 anti-rabbit IgG (Invitrogen) at 1:1000 dilutions for 30 min.

Techniques: Expressing, Knockdown

Journal: The Journal of Cell Biology

Article Title: Pluripotency state regulates cytoneme selectivity and self-organization of embryonic stem cells

doi: 10.1083/jcb.202005095

Figure Lengend Snippet: iGluR activity, but not LRP6 overexpression, control cell polarization to Wnt3a beads, and pESC–TSC approximation. (A and B) Representative images of pESCs stained with antibodies against LRP6 (A) and β-catenin (B), and with phalloidin (F-actin). Inserts are magnification of boxes, contrast enhanced for clarity. Scale bars, 20 µm for larger images, 5 µm for inserts. (C) The percentage of cytonemes positive for LRP6 or β-catenin in pESCs. n = 62 cells. (D) Lrp6 RNA expression levels in control (CNTRL) pESCs or pESCs transiently overexpressing LRP6-eGFP (LRP6 OE), presented as fold-change to CNTRL pESCs. Bars are mean of n = 3 experiments. Error bars are SEM. Statistical significance calculated by unpaired two-sided t test: ***, P < 0.001. (E) Representative images of CNTRL or LRP6-eGFP overexpressing pESCs, stained with antibodies against LRP6. BF is brightfield. Yellow arrowhead indicates high levels of LRP6 in the cytoneme. Images are maximum-intensity projections presented at equal intensity range to allow comparison between panels. Scale bars, 20 µm. (F) The percentage of reactive interactions (defined in ) between ESCs, CNTRL pESCs, pESCs overexpressing LRP6 (LRP6 OE), or pESCs treated with 100 µM kainate and TSCs. n ≥ 58 cells pooled from ≥3 independent experiments. Statistical significance calculated by multiple Fisher’s exact two-sided tests: ****, P < 0.0001. (G) Representative images of ESCs contacting a Wnt3a bead at the base of the cytonemes, stained with antibodies against LRP6 or β-catenin (cyan) and GluA3, GluA4, GluK1, and GluK3. Bead is black sphere in brightfield (BF) panel and is highlighted with a dashed yellow circle. Scale bars, 10 µm. (H and I) The percentage of control ESCs (CTRL, blue), 10 µM CNQX-treated ESCs (green), pESCs (CTRL, orange), or pESCs treated with 100 µM kainate (pink) with polarized presentation of LRP6 (H) or β-catenin (I) upon Wnt3a bead contact. n ≥ 41 cells. Further quantification is shown in . Statistical significance calculated by multiple Fisher’s exact two-sided tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (J) The percentage of ESCs (blue) or pESCs (orange) with polarized distribution of both Wnt/β-catenin pathway components and GluA3, GluA4, GluK1, or GluK3. n ≥ 26 cells. Statistical significance calculated by multiple Fisher’s exact two-sided tests: **, P < 0.01. (K and L) Representative frames of a time course live-cell imaging experiment showing a pESC (magenta) treated with 100 µM kainate contacting a TSC (green) through a cytoneme, approaching it, and then separating (K). Time is minutes, and yellow dashed line indicates distance between cells. Scare bar, 20 µm. Plot on L indicates pESC-TSC distance over time (cell in K only). Arrows point to distance at initial contact (X c ) and distance at 50 min after initial cytoneme-mediated contact (X c+50 ). (M) The difference in distance between CNTRL pESCs (orange) or 100 µM kainate–treated pESCs (KA, pink) and TSCs at the initial cytoneme-mediated contact (X c ) or 50 min after contact (X c+50 , Δ Distance = X c+50 – X c ). Bars are mean of n ≥ 58 cells pooled from ≥3 experiments. Error bars are SEM. Asterisks indicate statistical significance calculated by unpaired two-sided t test: **, P < 0.01.

Article Snippet: The antibodies used were anti-α-tubulin (YL1/2; rat; ab6160; Abcam), anti–β-catenin (mouse; #610154; BD Transduction), anti-LRP6 (EPR2423(2); rabbit; ab134146; Abcam), anti-NANOG (rabbit; RCAB002P-F; Reprocell), anti-OCT3/4 (mouse; #611202; BD Transduction), anti-EOMES (rabbit; ab183991; Abcam), anti-GriA3 (mouse; MAB5416; Sigma-Aldrich), anti-GriA4 (rabbit; AB1508; Sigma-Aldrich), anti-GriK1 (rabbit; AGC-008; Alomone Laboratories), anti-GriK3 (rabbit; AGC-040; Alomone Laboratories), anti–N-cadherin (mouse; #33-3900; Thermo Fisher Scientific), anti–E-cadherin (DECMA-1; rat; ab11512; Abcam), anti-GFP (chicken; GFP-1020; Aves), anti-mCherry (goat; #200-101-379; Rockland), and AF488, AF555, or AF647-conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Activity Assay, Over Expression, Staining, RNA Expression, Live Cell Imaging

Journal: The Journal of Cell Biology

Article Title: Pluripotency state regulates cytoneme selectivity and self-organization of embryonic stem cells

doi: 10.1083/jcb.202005095

Figure Lengend Snippet: Primers used for RT-qPCR

Article Snippet: The antibodies used were anti-α-tubulin (YL1/2; rat; ab6160; Abcam), anti–β-catenin (mouse; #610154; BD Transduction), anti-LRP6 (EPR2423(2); rabbit; ab134146; Abcam), anti-NANOG (rabbit; RCAB002P-F; Reprocell), anti-OCT3/4 (mouse; #611202; BD Transduction), anti-EOMES (rabbit; ab183991; Abcam), anti-GriA3 (mouse; MAB5416; Sigma-Aldrich), anti-GriA4 (rabbit; AB1508; Sigma-Aldrich), anti-GriK1 (rabbit; AGC-008; Alomone Laboratories), anti-GriK3 (rabbit; AGC-040; Alomone Laboratories), anti–N-cadherin (mouse; #33-3900; Thermo Fisher Scientific), anti–E-cadherin (DECMA-1; rat; ab11512; Abcam), anti-GFP (chicken; GFP-1020; Aves), anti-mCherry (goat; #200-101-379; Rockland), and AF488, AF555, or AF647-conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Sequencing

Journal: eNeuro

Article Title: Slow NMDA-Mediated Excitation Accelerates Offset-Response Latencies Generated via a Post-Inhibitory Rebound Mechanism

doi: 10.1523/ENEURO.0106-19.2019

Figure Lengend Snippet: Primary and secondary antibodies used for immunocytochemistry

Article Snippet: VGLUT3 , Peptide (C)ELNHEAFVSPRKK, corresponding to amino acid residues 533–545 of rat VGLUT3 (accession Q7TSF2); cytoplasmic, C terminus , Alomone Labs , AGC-037 , Rabbit , 1:300.

Techniques: Clone Assay, Recombinant, Purification, Isolation, Strep-tag

Journal: The Journal of Neuroscience

Article Title: GluA2 Trafficking Is Involved in Apoptosis of Retinal Ganglion Cells Induced by Activation of EphB/EphrinB Reverse Signaling in a Rat Chronic Ocular Hypertension Model

doi: 10.1523/JNEUROSCI.4376-14.2015

Figure Lengend Snippet: Increase in p-GluA2 protein levels in COH rat retinae. A, Representative immunoblots showing the changes in p-GluA2 and GluA2 protein expression in sham-operated (Ctr) and COH retinal extracts at different postoperational times (G1d, G3d, G1w, G2w, G3w, G4w, and G6w). B, Bar chart shows that the average p-GluA2/GluA2 ratio steadily increased with postoperational time. All of the data are normalized to Ctr. n = 6 for all groups. *p < 0.05, **p < 0.01, and ***p < 0.001 vs Ctr. C, Co-IP experiments showing the interactions between GluA2 and p-src, GluA2 and ephrinB2 in normal retinae and retinae with COH. Bands of p-src at the location corresponding to 65 kDa (top) and bands of ephrinB2 at 37 kDa (bottom) were detected in the immunoprecipitates derived using the antibody against GluA2 both in normal and COH retinal extracts (G2w). D, Co-IP experiments showing the interactions of phosphorylated tyrosine with GluA2, and with ephrinB2 in normal retinae and retinae with COH. A band of GluA2 at 110 kDa (top) and of ephrinB2 at 37 kDa (bottom) were detected in the immunoprecipitates derived using a phosphorylated antibody directly against tyrosine in both normal and COH retinal extracts.

Article Snippet: The sections were blocked for 2 h in 6% normal donkey serum, 1% bovine serum (for EphB1 immunostaining, adding 0.2% Triton X-100), dissolved in PBS at room temperature, and then incubated with the following primary antibodies at 4°C for 48 h: monoclonal mouse anti-EphB1 (1:50 dilution; R&D Systems), polyclonal goat anti-EphB2 (1:50 dilution; R&D Systems) or polyclonal rabbit anti-EphB2 (1:300 dilution; Santa Cruz Biotechnology), polyclonal goat anti-ephrinB1 (1:50 dilution; R&D Systems), polyclonal goat anti-ephrinB2 (1:50 dilution; R&D Systems), polyclonal rabbit anti-glutamine synthetase (GS; 1:2000 dilution; Abcam), polyclonal goat anti-CTB (1:2000 dilution; List Biological Laboratories), monoclonal mouse anti-GFAP (1:400 dilution; Sigma-Aldrich), monoclonal mouse anti-Brn-3a (1:100 dilution; Santa Cruz Biotechnology), and polyclonal rabbit anti-GluA2 (1:200 dilution; Alomone Labs).

Techniques: Western Blot, Expressing, Co-Immunoprecipitation Assay, Derivative Assay

Journal: The Journal of Neuroscience

Article Title: GluA2 Trafficking Is Involved in Apoptosis of Retinal Ganglion Cells Induced by Activation of EphB/EphrinB Reverse Signaling in a Rat Chronic Ocular Hypertension Model

doi: 10.1523/JNEUROSCI.4376-14.2015

Figure Lengend Snippet: Intravitreal injection of PP2 reduces p-src and p-GluA2 levels in EphB2-Fc-injected retinae and retinae with COH. A, Representative immunoblots showing the changes in protein levels of p-src, src, and p-GluA2; and in levels of GluA2 in Ctr-Fc-injected retinae, EphB2-Fc-injected retinae, and retinae with COH (G2w), with or without the addition of PP2. B, Bar chart summarizing the average ratios of p-src/src and p-GluA2/GluA2 under different conditions. All of the data are normalized to control (Ctr-Fc). n = 6 for all the groups.

Article Snippet: The sections were blocked for 2 h in 6% normal donkey serum, 1% bovine serum (for EphB1 immunostaining, adding 0.2% Triton X-100), dissolved in PBS at room temperature, and then incubated with the following primary antibodies at 4°C for 48 h: monoclonal mouse anti-EphB1 (1:50 dilution; R&D Systems), polyclonal goat anti-EphB2 (1:50 dilution; R&D Systems) or polyclonal rabbit anti-EphB2 (1:300 dilution; Santa Cruz Biotechnology), polyclonal goat anti-ephrinB1 (1:50 dilution; R&D Systems), polyclonal goat anti-ephrinB2 (1:50 dilution; R&D Systems), polyclonal rabbit anti-glutamine synthetase (GS; 1:2000 dilution; Abcam), polyclonal goat anti-CTB (1:2000 dilution; List Biological Laboratories), monoclonal mouse anti-GFAP (1:400 dilution; Sigma-Aldrich), monoclonal mouse anti-Brn-3a (1:100 dilution; Santa Cruz Biotechnology), and polyclonal rabbit anti-GluA2 (1:200 dilution; Alomone Labs).

Techniques: Injection, Western Blot

Journal: The Journal of Neuroscience

Article Title: GluA2 Trafficking Is Involved in Apoptosis of Retinal Ganglion Cells Induced by Activation of EphB/EphrinB Reverse Signaling in a Rat Chronic Ocular Hypertension Model

doi: 10.1523/JNEUROSCI.4376-14.2015

Figure Lengend Snippet: Changes in membrane GluA2 protein expression in retinae with COH. A, Representative immunoblots showing the changes in GluA2 protein levels in the cell membrane component (m-GluA2) in sham-operated (Ctr) and COH retinal extracts at different postoperational times (G1d, G3d, G1w, G2w, G3w, G4w, and G6w). B, Bar chart summarizing the average densitometric quantification of immunoreactive bands of m-GluA2 at different times. All of the data are normalized to Ctr. n = 6. *p < 0.05 and **p < 0.01 vs Ctr.

Article Snippet: The sections were blocked for 2 h in 6% normal donkey serum, 1% bovine serum (for EphB1 immunostaining, adding 0.2% Triton X-100), dissolved in PBS at room temperature, and then incubated with the following primary antibodies at 4°C for 48 h: monoclonal mouse anti-EphB1 (1:50 dilution; R&D Systems), polyclonal goat anti-EphB2 (1:50 dilution; R&D Systems) or polyclonal rabbit anti-EphB2 (1:300 dilution; Santa Cruz Biotechnology), polyclonal goat anti-ephrinB1 (1:50 dilution; R&D Systems), polyclonal goat anti-ephrinB2 (1:50 dilution; R&D Systems), polyclonal rabbit anti-glutamine synthetase (GS; 1:2000 dilution; Abcam), polyclonal goat anti-CTB (1:2000 dilution; List Biological Laboratories), monoclonal mouse anti-GFAP (1:400 dilution; Sigma-Aldrich), monoclonal mouse anti-Brn-3a (1:100 dilution; Santa Cruz Biotechnology), and polyclonal rabbit anti-GluA2 (1:200 dilution; Alomone Labs).

Techniques: Expressing, Western Blot

Journal: The Journal of Neuroscience

Article Title: GluA2 Trafficking Is Involved in Apoptosis of Retinal Ganglion Cells Induced by Activation of EphB/EphrinB Reverse Signaling in a Rat Chronic Ocular Hypertension Model

doi: 10.1523/JNEUROSCI.4376-14.2015

Figure Lengend Snippet: Changes in membrane GluA2 protein expression in EphB2-Fc-injected retinae and retinae with COH. A–C, Immunofluorescence labeling showing GluA2 expression profiles (green) in rat retinal vertical slices taken from Ctr-Fc-injected (a1) and EphB2-Fc-injected (b1) retinae 2 weeks after the injection, and from retinae with COH in G2w (c1). a2, b2, and c2, Brn-3a-labeled images (red). a3, b3, and c3, Merged images. D, E, GluA2 expression profiles (green) in rat retinal slices taken from EphB2-Fc-injected retinae, obtained 2 weeks after the injection (Dd1), and from retinae with COH, obtained in G2w (Ee1), when these preparations were intravitreally preinjected with PP2 (100 μm, 2 μl) 3 d before the operation or EphB2-Fc injection. d2, e2, Brn-3a-labeled images (red). d3, e3, Corresponding merged images. Scale bar, 20 μm. F, Representative immunoblots showing the changes in protein levels of m-GluA2 in retinae obtained under different conditions. G, Bar chart showing the average densitometric quantification of immunoreactive bands of m-GluA2 under different conditions. Note that EphB2-Fc injection/IOP elevation induced a significant decrease in m-GluA2 protein levels; preinjection of PP2 reversed the changes. All of the data are normalized to Ctr-Fc. n = 6 for all groups. *p < 0.05 and **p < 0.01 vs Ctr-Fc.

Article Snippet: The sections were blocked for 2 h in 6% normal donkey serum, 1% bovine serum (for EphB1 immunostaining, adding 0.2% Triton X-100), dissolved in PBS at room temperature, and then incubated with the following primary antibodies at 4°C for 48 h: monoclonal mouse anti-EphB1 (1:50 dilution; R&D Systems), polyclonal goat anti-EphB2 (1:50 dilution; R&D Systems) or polyclonal rabbit anti-EphB2 (1:300 dilution; Santa Cruz Biotechnology), polyclonal goat anti-ephrinB1 (1:50 dilution; R&D Systems), polyclonal goat anti-ephrinB2 (1:50 dilution; R&D Systems), polyclonal rabbit anti-glutamine synthetase (GS; 1:2000 dilution; Abcam), polyclonal goat anti-CTB (1:2000 dilution; List Biological Laboratories), monoclonal mouse anti-GFAP (1:400 dilution; Sigma-Aldrich), monoclonal mouse anti-Brn-3a (1:100 dilution; Santa Cruz Biotechnology), and polyclonal rabbit anti-GluA2 (1:200 dilution; Alomone Labs).

Techniques: Expressing, Injection, Immunofluorescence, Labeling, Western Blot

Journal: The Journal of Neuroscience

Article Title: GluA2 Trafficking Is Involved in Apoptosis of Retinal Ganglion Cells Induced by Activation of EphB/EphrinB Reverse Signaling in a Rat Chronic Ocular Hypertension Model

doi: 10.1523/JNEUROSCI.4376-14.2015

Figure Lengend Snippet: Activation of EphB2/ephrinB2 reverse signaling induces GluA2-containing AMPA receptor trafficking. A, B, Representative traces showing the changes in amplitude of evoked EPSCs, mediated by AMPA receptors, recorded from four different RGCs in retinal slices at holding potentials of +40 and −60 mV with (A) or without (B) spermine in the pipette solution. The eyes were intravitreally injected respectively with control-Fc (Ctr-Fc) and EphB2-Fc (2 μl, 1 μg/μl), and the recordings were made 5–7 d after the injections. The current amplitudes were all normalized to that of Ctr-Fc at −60 mV. C, Bar chart showing the average ratios of current amplitudes at +40/−60 mV in RGCs obtained from Ctr-Fc- and EphB2-Fc-injected retinae with or without spermine in the pipette solution. *p < 0.05 vs Ctr-Fc.

Article Snippet: The sections were blocked for 2 h in 6% normal donkey serum, 1% bovine serum (for EphB1 immunostaining, adding 0.2% Triton X-100), dissolved in PBS at room temperature, and then incubated with the following primary antibodies at 4°C for 48 h: monoclonal mouse anti-EphB1 (1:50 dilution; R&D Systems), polyclonal goat anti-EphB2 (1:50 dilution; R&D Systems) or polyclonal rabbit anti-EphB2 (1:300 dilution; Santa Cruz Biotechnology), polyclonal goat anti-ephrinB1 (1:50 dilution; R&D Systems), polyclonal goat anti-ephrinB2 (1:50 dilution; R&D Systems), polyclonal rabbit anti-glutamine synthetase (GS; 1:2000 dilution; Abcam), polyclonal goat anti-CTB (1:2000 dilution; List Biological Laboratories), monoclonal mouse anti-GFAP (1:400 dilution; Sigma-Aldrich), monoclonal mouse anti-Brn-3a (1:100 dilution; Santa Cruz Biotechnology), and polyclonal rabbit anti-GluA2 (1:200 dilution; Alomone Labs).

Techniques: Activation Assay, Transferring, Injection

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