Journal: Frontiers in Cellular Neuroscience

Article Title: Pin1 Is Regulated by CaMKII Activation in Glutamate-Induced Retinal Neuronal Regulated Necrosis

doi: 10.3389/fncel.2019.00276

Figure Lengend Snippet: Primer sequence for real-time PCR.

Article Snippet: The membranes were blocked in TBS-T containing 5% non-fat milk in for 1 or 2 h, at RT, and then incubated with the following primary antibodies overnight at 4°C: Pin1 (1:1,000, Cell Signaling), p-CaMKII (1:1,000, Bioss), CaMKII (1:1,000, 12666-2-AP, Proteintech, Rosemont, IL, United States), NR1 (1:500, bs-23343R, Bioss), GluR1 (1:500, bs-10042R, Bioss), GAPDH (1:5,000, AF0006, Beyotime, Beijing, China).

Techniques: Sequencing

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

Journal: iScience

Article Title: A Neuroligin-1 mutation associated with Alzheimer’s disease produces memory and age-dependent impairments in hippocampal plasticity

doi: 10.1016/j.isci.2023.106868

Figure Lengend Snippet: Age-dependent loss of AMPAR response in HTZ Nlgn1 Thr271fs mice (A) Normal dendritic spine density in HTZ Nlgn1 Thr271fs mice. Representative Golgi-Cox images and quantitation of dendritic spines density in CA1 neurons of the hippocampus of adult (5–6 months) and old (24 months) mice are shown (adult: wild-type vs. HTZ unpaired t-test p = 0.921; old: wild-type vs. HTZ unpaired t-test p = 0.543. Adult mice: wild-type, 10.16 ± 1.03; HTZ, 10.29 ± 0.77. Old mice: wild-type, 8.41 ± 0.62; HTZ, 7.84 ± 0.65). Four to five independent animals per condition, 8–20 dendritic segments per animal. (B) Whole-cell voltage-clamp experiments in hippocampal CA1 neurons from wild-type and HTZ Nlg1 Thr271fs mice at 1–2 months. AMPAR and NMDAR EPSCs were recorded at −70 and +40 mV, respectively. (C) Normal NMDAR/AMPAR ratio in HTZ Nlgn1 Thr271fs mice at 1–2 months (unpaired t-test p = 0.784). Wild-type, n = 12; HTZ, n = 11. (D) Input-output curves in hippocampal CA1 neurons from 1 to 2 months old mice recorded in whole-cell voltage-clamp experiments at −70 mV holding potential (multiple unpaired t-test p = 0.569, p = 0.902, p = 0.836, p = 0.398, p = 0.672, p = 0.648, p = 0.982, p = 0.850, p = 0.757). Wild-type, n = 10; HTZ, n = 7. (E) Representative traces of AMPAR and NMDAR EPSCs recorded in 18-months old CA1 hippocampal neurons. AMPAR EPSC was selectively reduced in HTZ Nlg1 Thr271fs mice. (F) Increased NMDAR/AMPAR ratio in HTZ Nlgn1 Thr271fs mice at 18 months (unpaired T-test p = 0.045). Wild-type, n = 12; HTZ, n = 11. (G) Decreased Input-output currents in 18-months old HTZ Nlgn1 Thr271fs mice recorded in EPSC experiments at −70 mV (multiple unpaired t-test p = 0.069, p = 0.425, p = 0.289, p = 0.242, p = 0.112, p = 0.078, p = 0.090, p = 0.117, p = 0.032). Wild-type, n = 7; HTZ, n = 8. (H and I) Expression of synaptic components in the hippocampus of Nlgn1 Thr271fs mice in 12-months old (H) and 6-months old (I) individuals. ((H) Nlgn1, one-way ANOVA with Tukey’s post-hoc analysis. F (2.6) = 251.6, p < 0.0001, wild-type vs. HTZ p < 0.0001, wild-type vs. HMZ p < 0.0001, HTZ vs. HMZ p < 0.0001. Wild-type, 1 ± 0.04; HTZ, 0.517 ± 0.035; HMZ: 0.005 ± 0.002. Nlgn2, one-way ANOVA. F (2.6) = 0.199, p = 0.825. Wild-type 1 ± 0.061; HTZ 1.078 ± 0.060; HMZ 1.122 ± 0.0531. Nlgn3, one-way ANOVA with Holm-Sidak’s post-hoc analysis. F (2.6) = 287.2, p < 0.0001, wild-type vs. HTZ p = 0.208, wild-type vs. HMZ p = 0.0005, HTZ vs. HMZ p = 0.002. Wild-type 1 ± 0.09; HTZ 1.28 ± 0.10; HMZ 2.196 ± 0.107. PSD95, one-way ANOVA. F (2.6) = 0.44, p = 0.662. Wild-type 1 ± 0.085; HTZ 1.090 ± 0.136; HMZ 1.14 ± 0.047. GluA1, one-way ANOVA. F (2.6) = 2.76, p = 0.15. Wild-type 1 ± 0.06; HTZ 1.051 ± 0.09; HMZ 0.835 ± 0.027. GluA2, one-way ANOVA. F (2.6) = 0.068, p = 0.934. Wild-type 1 ± 0.15; HTZ 1.03 ± 0.046; HMZ 1.041 ± 0.044. (I) Nlgn1, one-way ANOVA with Tukey’s post-hoc analysis. F (2.6) = 105.2, p < 0.0001, wild-type vs. HTZ p = 0.006, wild-type vs. HMZ p < 0.0001, HTZ vs. HMZ p = 0.0012. Wild-type 1 ± 0.08; HTZ 0.468 ± 0.007; HMZ 0.001 ± 0.0010. Nlgn2, one-way ANOVA. F (2.6) = 0.37, p = 0.702. Wild-type 1 ± 0.03; HTZ 0.952 ± 0.091; HMZ 0.922 ± 0.051. Nlgn3, one-way ANOVA with Holm-Sidak’s post-hoc analysis. F (2.6) = 13.53, p = 0.0096, wild-type vs. HTZ p = 0.47, wild-type vs. HMZ p = 0.0177, HTZ vs. HMZ p = 0.0177. Wild-type 1 ± 0.20; HTZ 1.16 ± 0.10; HMZ 1.952 ± 0.13. PSD95, one-way ANOVA. F (2.6) = 0.091, p = 0.914. Wild-type 1 ± 0.059; HTZ 1.037 ± 0.082; HMZ 1.012 ± 0.032). The expression of Nlgn1, Nlgn2, Nlgn3, PSD95, GluA1 and GluA2 was analyzed with specific antibodies, as indicated. Scale bar, 10 μm ∗p < 0.05. Data are represented as mean ± SEM. See also Figure S4 .

Article Snippet: Western blot experiments of lysates containing equal-protein loading (20-40 μg per lysate) were performed using the following primary antibodies: mouse anti N-terminal Nlgn1 (1:1,000, Synaptic Systems, clone 4C12, Cat# 129 111, RRID: AB_887747 ); mouse anti C-terminal Nlgn1 (1:1,000, UC Davis/NIH NeuroMab Facility Cat# N97A/31, clone N97A/31, RRID: AB_2877345 ); mouse anti NL2 (1:1,000, Synaptic Systems, clone 5E6, Cat# 129 511, RRID: AB_2619813 ); mouse anti NL3 (1:1,000, UC Davis/NIH NeuroMab Facility, clone N110/29, Cat# N110/29, RRID: AB_2877346 ); mouse anti GluA1 (1:500, Santa Cruz Biotechnology, Cat# sc-55509, RRID: AB_629532 ); mouse anti GluA2 (1:1,000, UC Davis/NIH NeuroMab Facility, Cat# L21/32, RRID: AB_2877267 ); mouse anti-PSD-95 (1:1,000, Thermo Fisher Scientific, Cat# MA1-045, RRID: AB_325399 ); and mouse anti-β actin (1:5,000, Sigma-Aldrich, Cat# A5316, RRID: AB_476743 ).

Techniques: Quantitation Assay, Expressing

Journal: iScience

Article Title: A Neuroligin-1 mutation associated with Alzheimer’s disease produces memory and age-dependent impairments in hippocampal plasticity

doi: 10.1016/j.isci.2023.106868

Figure Lengend Snippet:

Article Snippet: Western blot experiments of lysates containing equal-protein loading (20-40 μg per lysate) were performed using the following primary antibodies: mouse anti N-terminal Nlgn1 (1:1,000, Synaptic Systems, clone 4C12, Cat# 129 111, RRID: AB_887747 ); mouse anti C-terminal Nlgn1 (1:1,000, UC Davis/NIH NeuroMab Facility Cat# N97A/31, clone N97A/31, RRID: AB_2877345 ); mouse anti NL2 (1:1,000, Synaptic Systems, clone 5E6, Cat# 129 511, RRID: AB_2619813 ); mouse anti NL3 (1:1,000, UC Davis/NIH NeuroMab Facility, clone N110/29, Cat# N110/29, RRID: AB_2877346 ); mouse anti GluA1 (1:500, Santa Cruz Biotechnology, Cat# sc-55509, RRID: AB_629532 ); mouse anti GluA2 (1:1,000, UC Davis/NIH NeuroMab Facility, Cat# L21/32, RRID: AB_2877267 ); mouse anti-PSD-95 (1:1,000, Thermo Fisher Scientific, Cat# MA1-045, RRID: AB_325399 ); and mouse anti-β actin (1:5,000, Sigma-Aldrich, Cat# A5316, RRID: AB_476743 ).

Techniques: Software

Journal: Molecular and cellular neurosciences

Article Title: Characterization of alternatively spliced isoforms of AMPA receptor subunits encoding truncated receptors.

doi: 10.1016/j.mcn.2007.10.008

Figure Lengend Snippet: Fig. 1. Identification of splice variants of AMPA receptor subunits in rat hippocampal and chicken retina cultures. (A) RT–PCR products of AMPA receptor subunit GluR1, amplified from hippocampal cultures, 14 DIV. Amplification was performed using primers that span the flip/flop region and part of TM4. In the upper panel, the cDNA molecules containing the flip/flop exons were partially digested with RsaI previous to amplification in order to facilitate the amplification of the variant lacking the flip/flop exons (GluRΔi/o). (B) RT–PCR amplification of GluR4 subunit from chick retinal cultures, 5 DIV, using primers that span the flip/flop region and part of TM4. (C) Comparison of the amplified nucleotide sequences encoding full-length and truncated isoforms of AMPA receptor GluR1, and of the corresponding translation products. The 5 amino acid sequence originated by the frameshift, before the stop codon, is indicated. Corresponding sequences for the other AMPA receptor subunits are: KRPVPSV (GluR2), TRPVL (GluR3) and TRRVP (GluR4). (D) Schematic representation of truncated isoforms of AMPA receptor subunit, lacking the flip/flop cassette, the fourth transmembrane domain and the C-terminus (box).

Article Snippet: Anti-GFP polyclonal antibody was purchased from MBL International Corporation (Woburn, USA), anti-PSD 95 antibody was from Affinity BioReagents (Golden, CO) and anti-GluR1 polyclonal antibody was from Tocris Bioscience (Missouri, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Variant Assay, Comparison, Sequencing

Journal: Molecular and cellular neurosciences

Article Title: Characterization of alternatively spliced isoforms of AMPA receptor subunits encoding truncated receptors.

doi: 10.1016/j.mcn.2007.10.008

Figure Lengend Snippet: Fig. 3. Saturation binding curve of [3H]AMPA in membranes of HEK 293 cells expressing GluR1, GluR1+GluR1Δi/o (1:1) or GluR1Δi/o. HEK 293 cells were transfected and cell membranes were isolated. The curves represent specific binding of [3H]AMPA, calculated as the difference between the total binding and the nondisplaceable binding, as a function of the concentration of free [3H]AMPA. There is no statistically significant difference between the curves for cells expressing GluR1 and expressing GluR1+GluR1Δi/o (p=0.18). BMAX and KD were determined using non- linear regression followed by one site binding hyperbola equation. Data are relative to three independent experiments done in triplicate. The recombi- nant protein levels were found to be similar in all conditions by Western blot analysis (not shown).

Article Snippet: Anti-GFP polyclonal antibody was purchased from MBL International Corporation (Woburn, USA), anti-PSD 95 antibody was from Affinity BioReagents (Golden, CO) and anti-GluR1 polyclonal antibody was from Tocris Bioscience (Missouri, USA).

Techniques: Binding Assay, Expressing, Transfection, Isolation, Concentration Assay, Western Blot

Journal: Molecular and cellular neurosciences

Article Title: Characterization of alternatively spliced isoforms of AMPA receptor subunits encoding truncated receptors.

doi: 10.1016/j.mcn.2007.10.008

Figure Lengend Snippet: Fig. 4. Cell surface expression and subcellular localization of AMPA receptors containing truncated GluR1. (A) Biotinylated cell surface proteins were purified from HEK 293 cells, transfected with GluR1 and GFP-GluR1Δi/o, and analyzed by Western blot with anti-GFP antibody. Additionally, cell surface proteins were purified from COS7 cells expressing both GluR1 and GluR1Δi/o, and probed with an anti-GluR1 N-terminal antibody (B). (C) Surface staining (in vivo) of GluR1 and GluR1Δi/o with anti-GluR1 N-terminal antibody, and total staining of GluR1 with anti-GluR1 C-terminal antibody in transfected COS7 cells. (D) Immunofluorescence staining in vivo with anti-GFP antibody of cell surface receptors in hippocampal neurons (18 DIV), transfected at 14 DIV with GFP-GluR1 and GFP-GluR1Δi/o. (E) Double immunostaining of GFP and PSD 95 in permeabilized hippocampal neurons transfected at 14 DIV with GFP-GluR1 and/or GFP-GluR1Δi/o. Merged images of labeling with anti-GFP and anti-PSD 95 antibodies show co-localization of the full-length subunit, but not of the truncated isoform, with the synaptic marker. The lower panel shows high-magnification images of the boxed regions of the images in the upper panel.

Article Snippet: Anti-GFP polyclonal antibody was purchased from MBL International Corporation (Woburn, USA), anti-PSD 95 antibody was from Affinity BioReagents (Golden, CO) and anti-GluR1 polyclonal antibody was from Tocris Bioscience (Missouri, USA).

Techniques: Expressing, Purification, Transfection, Western Blot, Staining, In Vivo, Immunofluorescence, Double Immunostaining, Labeling, Marker

Journal: Molecular and cellular neurosciences

Article Title: Characterization of alternatively spliced isoforms of AMPA receptor subunits encoding truncated receptors.

doi: 10.1016/j.mcn.2007.10.008

Figure Lengend Snippet: Fig. 5. Truncated isoforms of AMPA receptor subunits may be associated with neuronal protection against excitotoxic stimuli and increased neuronal activity. (A) Hippocampal neurons transfected at 5 DIV with GFP-GluR1 and GFP-GluR1Δi/o were incubated at 6 DIV with glutamate (125 μM), fixed and immunolabeled with anti-GFP antibody, 14 h later. The viability of GFP-expressing cells was evaluated by fluorescence microscopy using the fluorescent dye Hoechst 33342. Data are presented as average±S.E.M. of four experiments, performed in independent preparations, and are expressed as a percentage of cell death observed in hippocampal neurons transfected with GFP alone (taken as 100%). Statistical analysis was performed by one-way ANOVA followed by Bonferroni's Multiple Comparison Test: ⁎⁎pb0.01 for GFP-GluR1 versus GFP, when stimulated with glutamate; ⁎pb0.05 for GFP-GluR1 versus GFP and GFP- GluR1+GFP-GluR1Δi/o versus GFP-GluR1, in control conditions, and for GFP-GluR1+GFP-GluR1Δi/o versus GFP-GluR1, when stimulated with glutamate. (B) Nuclear morphology of hippocampal neurons 7 DIV transfected with GFP-GluR1 and co-transfected with GFP-GluR1 and GFP-GluR1Δi/o, labeled with anti-GFP, in the presence or in the absence of the glutamate stimulus (white arrows, transfected cells). (C) Expression of truncated AMPA receptor subunit GluR1 in hippocampal cultures stimulated at 14 DIV with glutamate (150 μM) for 20 min. Total RNA was extracted 4 h later, RT–PCR was performed using non- digested cDNA samples and the amplification products were analyzed in an agarose gel, isolated and sequenced. (D) Expression of AMPA receptor subunit GluR3 in the cortex and in the hippocampus of epileptic rats, sacrificed 24 h after kainate injection. Products were amplified by PCR of cDNA obtained from RNA isolated from the hippocampus and the cortex of epileptic rats, without digestion with RsaI to eliminate full-length transcripts. In the case of cDNA samples from the striatum, olfactory bulb, cerebellum, and brainstem, the samples were digested with RsaI to eliminate the full-length transcripts before PCR amplification. The bottom panel shows the RT–PCR products obtained using RNA isolated from control rats.

Article Snippet: Anti-GFP polyclonal antibody was purchased from MBL International Corporation (Woburn, USA), anti-PSD 95 antibody was from Affinity BioReagents (Golden, CO) and anti-GluR1 polyclonal antibody was from Tocris Bioscience (Missouri, USA).

Techniques: Activity Assay, Transfection, Incubation, Immunolabeling, Expressing, Fluorescence, Microscopy, Comparison, Control, Labeling, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Isolation, Injection