Journal: Annals of Translational Medicine

Article Title: The recycling of AMPA receptors/GABAa receptors is related to neuronal excitation/inhibition imbalance and may be regulated by KIF5A

doi: 10.21037/atm-22-4337

Figure Lengend Snippet: Expression of KIF5A, GluR2 and beta 2+3 subunits of gamma aminobutyric acid receptors (Gabrb2+3) in an in vivo model of seizures. (A) EEG results: There was no epileptic discharge in the Ctl group after the injection of saline; however, epileptic discharge was observed in the Sez group after the injection of PTZ. (B) Total protein expression: the gray level of the total protein expression bands was normalized with GAPDH, and the total protein expression levels of KIF5A, GluR2 and Gabrb2+3 in the hippocampus of the Sez group did not change significantly (n=6 in each group, vs. Ctl, P>0.05). (C) Surface protein expression: The gray level of the total protein expression bands was normalized with Sodium/potassium-transporting ATPase subunit alpha-1 (ATP1A1). In the Sez group, the expression of GluR2 on the surface increased significantly to 181.74%±14.44% ( vs. Ctl, # , P<0.01). Conversely, the expression of GluR2 on the surface decreased to 19.62%±8.01% ( vs. Ctl, # , P<0.01). KIF5A, kinesin superfamily proteins 5A; GluR2, glutamate receptors subunit-2; Ctl, control; Sez, seizures; PTZ, pentylenetetrazol.

Article Snippet: The primary antibodies used include anti-KIF5A antibody 1:1,000 (SANTA, sc-376452), anti-GluR2 antibody 1:1,000 (BOSTER, PB9205), and anti-GABAaR β2+3 antibody 1:500 (Bioss, bs-12066R).

Techniques: Expressing, In Vivo, Injection

Journal: Annals of Translational Medicine

Article Title: The recycling of AMPA receptors/GABAa receptors is related to neuronal excitation/inhibition imbalance and may be regulated by KIF5A

doi: 10.21037/atm-22-4337

Figure Lengend Snippet: Receptor recycling assay (IF). The recycling ratio of GluR2 was 0.30±0.05 in the Ctl group and 0.60±0.07 in the Mg 2+ -free group (7 cells per group, vs. Ctl, # , P<0.01), and the recycling ratio of Gabrb2+3 was 0.49±0.04 in Ctl group and 0.32±0.05 in the Mg 2+ -free group (7 cells per group, vs. Ctl, # , P<0.01) (×600). Scale Bar: 50 µm. IF, immunofluorescence; Ctl, control.

Article Snippet: The primary antibodies used include anti-KIF5A antibody 1:1,000 (SANTA, sc-376452), anti-GluR2 antibody 1:1,000 (BOSTER, PB9205), and anti-GABAaR β2+3 antibody 1:500 (Bioss, bs-12066R).

Techniques: Immunofluorescence

Journal: Annals of Translational Medicine

Article Title: The recycling of AMPA receptors/GABAa receptors is related to neuronal excitation/inhibition imbalance and may be regulated by KIF5A

doi: 10.21037/atm-22-4337

Figure Lengend Snippet: Interaction between KIF5A and GluR2 and Gabrb2+3 in the seizure model. (A) Co-ip results: The protein bands showed the co-ip levels of KIF5A, GluR2, and Gabrb2+3, and normalized with the protein levels of KIF5A. The GluR2 level of KIF5A pull-down in the hippocampi of the rats in the Sez group increased to 130.42%±53.24% (n=6 per, vs. Ctl, *, P<0.05). However, the Gabrb2+3 level of KIF5A decreased to 50.86%±5.33% in the Sez group (n=6 per group, vs. Ctl, # , P<0.01). (B) IF results: the Pearson’s correlation coefficients (PCC) of KIF5A/GluR2 was 0.40±0.19 in the Ctl group and 0.87±0.11 in the Mg 2+ -free solution group (n=6 per group, vs. Ctl, # , P<0.01) (×400). Scale Bar: 100 µm. (C) IF results: the PCC of KIF5A/Gabrb2+3 was 0.97±0.02 in the Ctl group and 0.32±0.11 in the Mg 2+ -free solution group (n=6 per group, vs. Ctl, # , P<0.01) (×400). Scale Bar: 100 µm. IF, immunofluorescence.

Article Snippet: The primary antibodies used include anti-KIF5A antibody 1:1,000 (SANTA, sc-376452), anti-GluR2 antibody 1:1,000 (BOSTER, PB9205), and anti-GABAaR β2+3 antibody 1:500 (Bioss, bs-12066R).

Techniques: Co-Immunoprecipitation Assay, Immunofluorescence

Journal: Toxicology Reports

Article Title: Development of a simple measurement method for GluR2 protein expression as an index of neuronal vulnerability

doi: 10.1016/j.toxrep.2014.12.014

Figure Lengend Snippet: AlphaLISA assay of GluR2 protein expression. (A) Principles of the AlphaLISA assay; biotinylated anti-GluR2 mouse monoclonal antibody binds streptavidin-coated alpha donor beads and anti-GluR2 rabbit polyclonal antibody binds anti-rabbit IgG-coated alpha acceptor beads. Both antibodies recognize GluR2, allowing the beads to come into close proximity. The excitation of the donor beads at 680 nm provokes the release of singlet oxygen molecules that trigger an energy transfer cascade in acceptor beads, resulting in a sharp emission peak at 615 nm. (B) AlphaLISA assay protocol.

Article Snippet: Rabbit anti-GluR2 pAb (BS3658) was purchased from Bioworld Technology, Inc. (St. Louis, MN, USA).

Techniques: Expressing

Journal: Toxicology Reports

Article Title: Development of a simple measurement method for GluR2 protein expression as an index of neuronal vulnerability

doi: 10.1016/j.toxrep.2014.12.014

Figure Lengend Snippet: GluR2 complex formation for AlphaLISA assays. (A) Cell lysates, anti-GluR2 antibodies, and AlphaLISA beads were incubated in microtubes without anti-rabbit IgG-coated alpha acceptor beads or without streptavidin-coated alpha donor beads. After incubation, bead fractions were separated by centrifugation at 15,000 rpm for 15 min. (B) Western blotting of fraction buffer with extra antibodies and extra GluR2; fraction beads bound with antibodies and GluR2. TNE buffer with cell lysis buffer was used as a control. (B) Western blotting of fraction , buffer with extra antibodies and extra GluR2 fraction , beads bound with antibodies and GluR2. TNE buffer with cell lysis buffer was used as a control.

Article Snippet: Rabbit anti-GluR2 pAb (BS3658) was purchased from Bioworld Technology, Inc. (St. Louis, MN, USA).

Techniques: Incubation, Centrifugation, Western Blot, Lysis

Journal: Toxicology Reports

Article Title: Development of a simple measurement method for GluR2 protein expression as an index of neuronal vulnerability

doi: 10.1016/j.toxrep.2014.12.014

Figure Lengend Snippet: Optimized assay conditions for detecting GluR2 expression. (A) Determination of optimum concentrations of biotinylated anti-GluR2 mouse monoclonal antibody, and (B) anti-GluR2 rabbit polyclonal antibody; (C) determination of optimum protein concentrations of cell lysates; rat primary cerebral cortical neurons were lysed in TNE buffer, and total protein abundance was quantified using bicinchoninic acid (BCA) protein assays; (D) enlarged view of (C); (E) time course of incubation: 1st incubation, binding of GluR2 protein and anti-GluR2 antibodies; 3rd incubation, binding of acceptor beads and anti-GluR2 rabbit polyclonal antibody. Data are expressed as the mean ± standard error of the mean ( n = 3).

Article Snippet: Rabbit anti-GluR2 pAb (BS3658) was purchased from Bioworld Technology, Inc. (St. Louis, MN, USA).

Techniques: Expressing, Incubation, Binding Assay

Journal: Toxicology Reports

Article Title: Development of a simple measurement method for GluR2 protein expression as an index of neuronal vulnerability

doi: 10.1016/j.toxrep.2014.12.014

Figure Lengend Snippet: Schematic representation of GluR2 protein and AlphaLISA assay. (A) GluR2 is a predicted six-transmembrane protein; (B) Schematics of the GluR2Δ1–169, 539–834 construct; numbers above the amino acid residues refer to residues of the full-length receptor; (C) measurement of purified GluR2 standard using AlphaLISA. Data are expressed as the mean ± the standard error of the mean ( n = 3). (D) Correlation between western blotting analyses and AlphaLISA assays. Quantitative western blotting analyses of GluR2 protein expression were performed using image J software. (E) Interference of the other proteins except GluR2 protein by means of the lysate of C6 cells. AlphaLISA was conducted using identical samples which contain the GluR2 standard and the lysates of C6 cells.

Article Snippet: Rabbit anti-GluR2 pAb (BS3658) was purchased from Bioworld Technology, Inc. (St. Louis, MN, USA).

Techniques: Construct, Purification, Western Blot, Expressing, Software

Journal: Toxicology Reports

Article Title: Development of a simple measurement method for GluR2 protein expression as an index of neuronal vulnerability

doi: 10.1016/j.toxrep.2014.12.014

Figure Lengend Snippet: Test compounds for the screening of GluR2 decrease.

Article Snippet: Rabbit anti-GluR2 pAb (BS3658) was purchased from Bioworld Technology, Inc. (St. Louis, MN, USA).

Techniques: Inhibition

Journal: Toxicology Reports

Article Title: Development of a simple measurement method for GluR2 protein expression as an index of neuronal vulnerability

doi: 10.1016/j.toxrep.2014.12.014

Figure Lengend Snippet: Changes in protein expression of GluR2 following long-term exposure to nitenpyram or TBT. (A) Cortical neurons were exposed to 0.1–100 μM NIT or 50 nM TBT for 9 days from 2 days in vitro (DIV) to 10 DIV, and GluR2 was then detected using western blotting. Quantitative analysis of GluR2 western blots were performed using ImageJ software, and GluR2 protein levels were normalized to those of β-actin. Data are expressed as the mean + standard error of the mean ( n = 4) * p < 0.05, ** p < 0.01 vs. DMSO. (B) Correlation between western blotting analyses and AlphaLISA assays. Quantitative western blotting analyses of GluR2 protein expression were performed using image J software.

Article Snippet: Rabbit anti-GluR2 pAb (BS3658) was purchased from Bioworld Technology, Inc. (St. Louis, MN, USA).

Techniques: Expressing, In Vitro, Western Blot, Software

Journal: PLoS Pathogens

Article Title: Mutant prion proteins increase calcium permeability of AMPA receptors, exacerbating excitotoxicity

doi: 10.1371/journal.ppat.1008654

Figure Lengend Snippet: (A) Hippocampal protein extracts (500 μg) from WT, FFI and CJD mice were incubated with uncoated magnetic beads (IP: no Ab) or magnetic beads coated with anti-PrP monoclonal antibody 94B4 (IP: PrP). The immunoprecipitated proteins were analyzed by western blot with anti-GluA1 or anti-GluA2 antibody or anti-PrP polyclonal antibody P45-66. The input is 20 μg of total proteins. Lanes 4 and 8 were left empty on purpose. This experiment is representative of three similar ones. (B) HeLa cells were co-transfected with plasmids encoding WT, FFI or CJD PrP-EGFP fusion protein, and the AMPAR subunit GluA2. After 48h, cells were fixed, permeabilized, stained with anti-GluA2 (red) antibody, and reacted with Hoechst 33258 (blue) to stain the nuclei. Scale bar 10 μm. (C, D) The fluorescent density of PrP and GluA2 on the cell surface was measured and background corrected (Corrected IntDen). Each bar indicates the mean ± SEM of 12–17 tranfected cells from three independent experiments. **p < 0.01, ****p < 0.0001 by one-way ANOVA, Tukey’s post hoc test.

Article Snippet: They were then co-incubated with rabbit polyclonal anti-GluA2 antibody (Synaptic System 182103, 1:200) or rabbit polyclonal anti-GluA4 antibody (Alomone AGC-019, 1:200) and guinea pig polyclonal anti-VGLUT1 antibody (Synaptic System 135304, 1:1000) for 72h at 4°C.

Techniques: Incubation, Magnetic Beads, Immunoprecipitation, Western Blot, Transfection, Staining

Journal: PLoS Pathogens

Article Title: Mutant prion proteins increase calcium permeability of AMPA receptors, exacerbating excitotoxicity

doi: 10.1371/journal.ppat.1008654

Figure Lengend Snippet: (A) Cerebellar protein extracts (500 μg) from Tg(WT), Tg(PG14) and PrP knockout (KO) mice were incubated with uncoated magnetic beads (IP: no Ab) or magnetic beads coated with anti-PrP monoclonal antibody 94B4 (IP: PrP). The immunoprecipitated proteins were analyzed by western blot with anti-GluA2 or anti-GluA4 antibody or anti-PrP polyclonal antibody P45-66. The input is 20 μg of total proteins. (B, C) The amounts of immunoprecipitated GluA2 (B) and GluA4 (C) were quantified by densitometry of western blots like in (A) and normalized on the amount of immunoprecipitated PrP. Data are the mean ± SEM of 3–4 replicates from three independent experiments; **p < 0.01 by Student’s t-test.

Article Snippet: They were then co-incubated with rabbit polyclonal anti-GluA2 antibody (Synaptic System 182103, 1:200) or rabbit polyclonal anti-GluA4 antibody (Alomone AGC-019, 1:200) and guinea pig polyclonal anti-VGLUT1 antibody (Synaptic System 135304, 1:1000) for 72h at 4°C.

Techniques: Knock-Out, Incubation, Magnetic Beads, Immunoprecipitation, Western Blot

Journal: PLoS Pathogens

Article Title: Mutant prion proteins increase calcium permeability of AMPA receptors, exacerbating excitotoxicity

doi: 10.1371/journal.ppat.1008654

Figure Lengend Snippet: (A) HeLa cells were co-transfected with plasmids encoding WT or PG14 PrP-EGFP fusion protein, and the AMPAR subunit GluA2. After 48h cells were fixed, permeabilized, stained with anti-GluA2 (red), and reacted with Hoechst 33258 (blue) to stain the nuclei. (B) The fluorescent density of GluA2 on the cell surface was measured and corrected for background (Corrected IntDen). Each bar indicates the mean ± SEM of 12 WT and 14 PG14 PrP-transfected cells from three independent experiments; *p<0.01 by Student’s t-test. (C) HeLa cells were co-transfected with WT or PG14 PrP and EGFP-tagged GluA4. After 48 h cells were fixed, permeabilized, stained with anti-PrP monoclonal antibody 98A3 (green), and reacted with Hoechst 33258 (blue) to stain the nuclei. (D) The fluorescent density of GluA4 on the cell surface was measured and background corrected. Each bar indicates the mean ± SEM of 6 WT and 6 PG14 PrP-transfected cells. (E) CGNs from WT and PG14 mice were fixed, permeabilized and immunostained with mouse monoclonal anti-GluA2 (green) and chicken polyclonal anti-MAP2 (red). Cells were reacted with Hoechst 33258 (blue) to stain the nuclei, and analyzed by confocal microscopy using sequential Z-stack acquisition mode and 3D reconstruction. The GluA2 signal on dendrites (F) and cell bodies (G) was analyzed by NIH ImageJ software. Data are the mean ± SEM of 18 WT cells (28 dendrites) and 18 PG14 cells (27 dendrites) from three independent experiments. (H-K) Brain sections of WT and PG14 mice were stained with anti-GluA2 (H) or GluA4 (J) (red) and anti-VGLUT1 (green) antibodies. The GluA2 (I) and GluA4 (K) in each cerebellar glomerulus were measured and expressed as the ratio between the area of the AMPA receptor subunit and VGLUT1 signals. The dotted line identifies a single glomerulus used for quantification. Signal outside the ROI has been cleared with ImageJ for clarity. Bars indicate the mean ± SEM of three mice per group (1098–1392 glomeruli). *p < 0.05 by Student’s t-test. Scale bars 20 μm in A, C and E; and 125 μm in H and J.

Article Snippet: They were then co-incubated with rabbit polyclonal anti-GluA2 antibody (Synaptic System 182103, 1:200) or rabbit polyclonal anti-GluA4 antibody (Alomone AGC-019, 1:200) and guinea pig polyclonal anti-VGLUT1 antibody (Synaptic System 135304, 1:1000) for 72h at 4°C.

Techniques: Transfection, Staining, Confocal Microscopy, Software