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

Article Title: Collybistin activation by GTP-TC10 enhances postsynaptic gephyrin clustering and hippocampal GABAergic neurotransmission

doi: 10.1073/pnas.1309078110

Figure Lengend Snippet: TC10 stimulates Cb-dependent gephyrin microcluster formation in COS-7 cells. (A1–A5) Images of COS-7 cells transfected as indicated. GFP-gephyrin accumulates in large cytoplasmic aggregates when expressed alone (A1) or together with Myc-SH3(+)CbII (A3) or HA-TC10 (A4). In the presence of Myc-ΔSH3CbII (A2), GFP-gephyrin forms microclusters at the plasma membrane. Similarly, HA-TC10 and Myc-SH3(+)CbII jointly trigger GFP-gephyrin microcluster formation (A5). (Scale bar, 10 µm.) (B) Schematic representation of the Cb splice variants and mutants, as well as of the TC10 WT and mutants, used in this study. (C1) Percentages of GFP-gephyrin (co)-transfected cells classified as displaying GFP-gephyrin microclusters (>50 puncta per cell; n = 3 independent transfections, n = 321–428 cells). Significance levels compared with cells transfected with GFP-gephyrin only (gray bar) are shown within the bars. (C2) Total numbers of GFP-gephyrin clusters and aggregates from images of transfected COS-7 cells (n = 14–34 transfected cells per transfection condition). Significance indicated as in C1. (C3) Gephyrin puncta counted in C2 were binned according to their size (n = 14–34 cells). (Insets) Relative fractions of small microclusters (0.05–0.2 µm2; Left) and aggregates (>1 µm2; Right). (D) Percentages of microclusters (0.04–0.4 µm2) per cell in COS-7 cells transfected as indicated (n = 6–34 cells). Statistical significance was tested between the conditions without coexpression of HA-TC10 (first four columns) and those in the presence of TC10 (WT, CA, or DN).

Article Snippet: Neurons were transfected at DIV 4 using the CalPhos mammalian transfection kit (Clontech).

Techniques: Transfection

Journal: The Journal of Biological Chemistry

Article Title: Cytochrome P450 2C Epoxygenases Mediate Photochemical Stress-induced Death of Photoreceptors *

doi: 10.1074/jbc.M113.507152

Figure Lengend Snippet: Expression of CYP2C is inducible by light and targeted knockdown of CYP2C55 rescues light-induced cell death. Deduced amino acids of encoded human CYP2C9 and mouse CYP2C55 and CYP2C29 enzymes were aligned using Clustal Omega. Rectangular boxes represent key conserved residues for catalytic activity (gray), ligand binding (black), and heme stabilization (dashed) (see Ref. 90) (A). 661W cells on 6-well plates with/without sulfaphenazole pretreatment for 2 h at 37 °C were exposed to light (L) or remained in the dark (D). Expression of CYP2C proteins was detected from prepared cell lysates by Western blotting using anti-human CYP2C9 polyclonal antibody that cross-reacts with mouse CYP2C isozymes. The housekeeping gene β-actin was used as a loading control. Relative expression is presented by normalization with the densitometric ratio of CYP2C/β-actin in dark control cells (B). 661W cells at 50% confluence were transfected with Silencer Select predesigned siRNAs targeted to mouse CYP2C55 or CYP2C29 or mock-transfected with the scrambled control (Con.) oligos (n = 4/sample group). Cell lysates were prepared at 48 h after transfection to assess silencing efficiency by Western blotting. The densitometric ratio of CYP2C55 or CYP2C29/β-actin is presented (C). Cellular ATP indicative of cell viability was measured after light exposure (n = 4/sample group) (D). **, p < 0.01; *, p < 0.05; n.s., not significant. RLU, relative light units. In B, bars represent the relative ratio of densitometry (CYP2C/β-actin) from one representative experiment. In C, bars represent the absolute ratio of densitometry (siCYP2C55 or siCYP2C29/β-actin).

Article Snippet: Generation of Stably Transfected CYP2C9-GFP Cell Lines in 661W Cells 661W cells at 50% confluence on 96-well plates were transfected with pCMV6-CYP2C9-GFP PrecisionShuttle mammalian expression vector (0.2 μg/well) using Turbofectin 8.0 (OriGene).

Techniques: Expressing, Activity Assay, Ligand Binding Assay, Western Blot, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Cytochrome P450 2C Epoxygenases Mediate Photochemical Stress-induced Death of Photoreceptors *

doi: 10.1074/jbc.M113.507152

Figure Lengend Snippet: Stable expression of functional human CYP2C9-GFP fusion protein enhances cellular sensitivity to light. pCMV6-CYP2C9-GFP stably transfected 661W cells were examined by confocal microscopy showing the bright field and its switch to FITC mode to assess homogeneity of GFP-positive cells. The arrow indicates expression of CYP2C9-GFP on the surface of the cell body (A). The enzymatic activity of CYP2C9-GFP fusion protein with/without sulfaphenazole (10 μm) pretreatment was examined by a cell-based luminogenic assay that generates luminogenic signal from CYP2C9-catalyzed oxidation of the selective substrate luciferin-H. Wild-type (WT) 661W cells were controls. n = 3/luciferin-H concentration. ***, p < 0.001; *, p < 0.05 versus CYP2C9-GFP + SFZ (B). pCMV6-CYP2C9-GFP stably transfected 661W cells with/without pretreatment with sulfaphenazole (10 μm) (n = 12/treatment or non-treatment group) and wild-type cells (n = 24) were exposed to light for 4 h. Cellular ATP indicative of cell viability was measured after light (L) exposure (C). n.s., not significant. Error bars represent means ± S.D. RLU, relative light units; D, dark.

Article Snippet: Generation of Stably Transfected CYP2C9-GFP Cell Lines in 661W Cells 661W cells at 50% confluence on 96-well plates were transfected with pCMV6-CYP2C9-GFP PrecisionShuttle mammalian expression vector (0.2 μg/well) using Turbofectin 8.0 (OriGene).

Techniques: Expressing, Functional Assay, Stable Transfection, Transfection, Confocal Microscopy, Activity Assay, Concentration Assay