Journal: Frontiers in Neuroinformatics

Article Title: Maximum likelihood-based estimation of diffusion coefficient is quick and reliable method for analyzing estradiol actions on surface receptor movements

doi: 10.3389/fninf.2023.1005936

Figure Lengend Snippet: The effect of E2 treatment on the diffusion coefficient of GluR2-AMPAR molecules in the soma’s plasma membranes of dPC12, live-cell. The E2 treatments were carried out by the concentration of 100 pM (A,B) and 100 nM (C,D) . Both the mean square displacement (MSD) (A,C) and the maximum likelihood-based estimation (MLE) (B,D) methods were used for further analysis to obtain the diffusion coefficients from the recorded trajectories. The graphs represent the groups as mean and SD values. The probability values of significant differences calculated by Kolmogorov–Smirnov test (* p < 0.05) and the number of trajectories in each group are also shown.

Article Snippet: Before single-molecule imaging, dPC12 were incubated with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of rat GluR2 (1:100, Alomone Labs Cat #: AGC-005-AG) in dRPMI cell culture medium at 37°C for 6 min. During the measurement period of ATTO 488-GluR2-AMPAR, 20–30 image series were recorded.

Techniques: Diffusion-based Assay, Concentration Assay

Journal: Frontiers in Neuroinformatics

Article Title: Maximum likelihood-based estimation of diffusion coefficient is quick and reliable method for analyzing estradiol actions on surface receptor movements

doi: 10.3389/fninf.2023.1005936

Figure Lengend Snippet: The trajectories length distribution from GluR2-AMPAR molecules in the soma’s plasma membranes of dPC12, live-cell in control state and after administration of 100 nM E2.

Article Snippet: Before single-molecule imaging, dPC12 were incubated with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of rat GluR2 (1:100, Alomone Labs Cat #: AGC-005-AG) in dRPMI cell culture medium at 37°C for 6 min. During the measurement period of ATTO 488-GluR2-AMPAR, 20–30 image series were recorded.

Techniques: Control

Journal: Aging (Albany NY)

Article Title: FGF22 promotes generation of ribbon synapses through downregulating MEF2D

doi: 10.18632/aging.103042

Figure Lengend Snippet: FGF22 depletion reduces ribbon synapse number in vivo. AAV-shFGF22 and AAV-FGF22 were administrated to mouse cochlea to deplete or overexpress FGF22 in hair cells, respectively. Co-administration of AAV-shMEF2D with AAV-shFGF22 was also performed to assess the regulatory relationship between FGF22 and MEF2D in hair cells and ribbon synapses. Similarly, co-administration of AAV-MEF2D with AAV-FGF22 was performed, also to assess the regulatory relationship between FGF22 and MEF2D in hair cells and ribbon synapses. ( A , B ) TUNEL staining was performed on cochlea, showing by representative images ( A ), and by quantification ( B ). ( C , D ) The ribbon synapses were determined by co-staining for CtBP2 (in green) and GluR2&3 (in red). Cochlear ribbon synapse number was assessed, shown by representative images ( C ), and by quantification ( D ). *p<0.05. NS: non-significant. N=5. Scale bar is 20μm.

Article Snippet: Primary antibodies are rabbit anti-CtBP2 polyclonal antibody (ab128871, 1:100, Abcam, Cambridge, MA, USA), rabbit anti-GluR2&3 polyclonal antibody (2mg/mL, Fisher Scientific, CA, USA) and rabbit anti-Myosin7a (1:200, ab3481, Abcam).

Techniques: In Vivo, TUNEL Assay, Staining

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