Journal: Biochimica et biophysica acta

Article Title: Modulation of α(2C) adrenergic receptor temperature-sensitive trafficking by HSP90.

doi: 10.1016/j.bbamcr.2010.11.020

Figure Lengend Snippet: Fig. 6. The effects of GRP94 overexpression and proteasomal inhibition on the α2C-AR temperature-sensitive trafficking. A. HEK293T cells were co-transfected with α2C-AR and pcDNA 3.1 (left columns) or GRP94 (right columns). The cell surface α2C-AR levels in cells maintained at 37 °C (black columns) or at 30 °C for 18 h (white columns) were determined in intact cells as above. mean±SD, n=9 from three independent transfections. B. α2C-AR transfected HEK293T cells were incubated with DMSO (vehicle, 0.1%), MG132 (10 μM) or lactacystin (5 μM) and subsequently exposed to 30 °C for 18 h or maintained at 37 °C. The α2C-AR plasma membrane levels were determined in intact cells. n=9 from three independent transfections. Fig. 5. Interactions between α2C-AR and HSP90 in HEK293T cells. A. HEK293T cells were transfected with empty vector pcDNA 3.1, or HA-tagged α2C-AR and 3×HA tagged α2B- AR (3 μg/10 cm2 plate each) and 6 h later the medium was changed to DMEM without FBS, followed by incubation at 30 °C or at 37 °C for the subsequent 18 h. Some plates were treated with macbecin (5 μM). Subsequently, the cells were solubilized and immunoprecipitated with HA antibody as described under Materials and methods. The HA immunoprecipitates (20 μg/lane) were separated by 10% SDS-Page and the HSP90 levels were revealed by Western-blotting. The experiment shown is representative from three independent transfections. Similar results were obtained in case of α2C-AR using GFP-tagged receptor and GFP antibody. B. Quantification of the data presented in A. mean±SD, n=3, *indicate statistical significant differences compared with control cells (pb0.05).

Article Snippet: Lactacystin and MG132 were from Tocris.

Techniques: Over Expression, Inhibition, Transfection, Incubation, Clinical Proteomics, Membrane, Plasmid Preparation, Immunoprecipitation, SDS Page, Western Blot, Control

Journal: Epilepsia

Article Title: Participation of metabotropic glutamate receptors in pentetrazol-induced kindled seizure.

doi: 10.1111/j.1528-1167.2010.02764.x

Figure Lengend Snippet: Figure 4. Effects of AIDA, (2R,4R)-APDC and L-AP4 on pentetrazol-induced kindled seizures in mice. Each col- umn represents the mean ± SEM (n = 8). *, **Significantly different from the control group at p < 0.05 and p < 0.01, respectively. Epilepsia ILAE

Article Snippet: As the mGluR agonists and antagonists, (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; Tocris Cookson Ltd., Bristol, UK), (2R,4R)-4-aminopyrrolidine2,4-dicarboxylate ((2R,4R)-APDC; Tocris Cookson), L-(+)2-amino-4-phosphonobutyric acid (L-AP4; Tocris Cookson), (RS)-3,5-dihydroxyphenylglycine ((RS)-3,5-DHPG; Tocris Cookson), (2S)-a-ethylglutamic acid (EGLU; Tocris Cookson), and (RS)-a-methyl-4-phosphonophenylglycine (MPPG; Tocris Cookson) were used.

Techniques: Control

Journal: Epilepsia

Article Title: Participation of metabotropic glutamate receptors in pentetrazol-induced kindled seizure.

doi: 10.1111/j.1528-1167.2010.02764.x

Figure Lengend Snippet: Figure 5. Effects of simultaneous use of mGluR agonists and antagonists on pentetrazol-induced kindled seizures. (A) Separate and simultaneous use of AIDA and (2R,4R)-APDC, (B) separate and simultaneous use of AIDA and L-AP4, and (C) separate and simultaneous use of (2R,4R)-APDC and L-AP4. Each column represents the mean ± SEM (n = 8). Cont, control; Comb, combined use of mGluR agonist and antagonist. *, **Significantly different from mGluR agonist- or antagonist- treated groups at p < 0.05 and p < 0.01, respectively. Epilepsia ILAE

Article Snippet: As the mGluR agonists and antagonists, (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; Tocris Cookson Ltd., Bristol, UK), (2R,4R)-4-aminopyrrolidine2,4-dicarboxylate ((2R,4R)-APDC; Tocris Cookson), L-(+)2-amino-4-phosphonobutyric acid (L-AP4; Tocris Cookson), (RS)-3,5-dihydroxyphenylglycine ((RS)-3,5-DHPG; Tocris Cookson), (2S)-a-ethylglutamic acid (EGLU; Tocris Cookson), and (RS)-a-methyl-4-phosphonophenylglycine (MPPG; Tocris Cookson) were used.

Techniques: Control

Journal: Epilepsia

Article Title: Participation of metabotropic glutamate receptors in pentetrazol-induced kindled seizure.

doi: 10.1111/j.1528-1167.2010.02764.x

Figure Lengend Snippet: Figure 8. Effects of (RS)-3,5-DHPG, EGLU, and MPPG on the inhibition of pentetrazol-induced kindled seizures caused by L-AP4. Each column represents the mean ± SEM (n = 8). ** Significantly different from the control group at p < 0.01. #Significantly different from L-AP4 (20 nmol/site, i.c.v.)–treated groups at p < 0.05. Epilepsia ILAE

Article Snippet: As the mGluR agonists and antagonists, (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; Tocris Cookson Ltd., Bristol, UK), (2R,4R)-4-aminopyrrolidine2,4-dicarboxylate ((2R,4R)-APDC; Tocris Cookson), L-(+)2-amino-4-phosphonobutyric acid (L-AP4; Tocris Cookson), (RS)-3,5-dihydroxyphenylglycine ((RS)-3,5-DHPG; Tocris Cookson), (2S)-a-ethylglutamic acid (EGLU; Tocris Cookson), and (RS)-a-methyl-4-phosphonophenylglycine (MPPG; Tocris Cookson) were used.

Techniques: Inhibition, Control

Journal: Chemistry & biology

Article Title: Inhibition of Wnt/β-catenin signaling by p38 MAP kinase inhibitors is explained by cross-reactivity with casein kinase Iδ/ɛ.

doi: 10.1016/j.chembiol.2011.01.015

Figure Lengend Snippet: Figure 4. Inhibition of CKId/3 Inhibits rmWnt-3a-Induced Phosphorylation of Human Dishevelled 2 and Blocks Signaling Through the Wnt/ b-Catenin Pathway (A) U2OS-EFC cells were treated with increasing concentrations of the CKI inhibitors IC261 (upper panel) and D4476 (lower panel) in the presence of 10 nM rmWnt-3a for 1 hr. Cell lysates were immunoblotted for human Dishevelled-2 (hDvl2) and b-actin. As a control for Wnt/b-catenin pathway inhibition, cells were cotreated with 10 nM rmWnt-3a and 1 mg/ml human Wnt inhibitory factor-1 (WIF1). See also Figure S3. (B) U2OS-EFC cells were treated with increasing concentrations of IC261 and D4476 in the presence of 10 nM rmWnt-3a for 3 hr, followed by measurement of b-galactosidase activity. Data are presented as mean ± SEM. See also Figures S4 and S5.

Article Snippet: D4476 and SB239063 were obtained from Tocris Cookson Inc. (St. Louis, MO).

Techniques: Inhibition, Phospho-proteomics, Control, Activity Assay

Journal: Endocrinology

Article Title: The calcium signaling pathway regulates leydig cell steroidogenesis through a transcriptional cascade involving the nuclear receptor NR4A1 and the steroidogenic acute regulatory protein.

doi: 10.1210/en.2012-1767

Figure Lengend Snippet: FIG. 1. Blocking internal Ca2 release impairs steroidogenesis. A, Intracellular Ca2 from MA-10 Leydig cells was quantified after a 15-min treatment with vehicle (DMSO, white bar), Fsk (10 M, black bar), or Fsk plus ryanodine (Rya) (150 M, gray bar). The assay was performed in duplicate in three independent experiments. **, P 0.02. B, ELISA was used to quantify progesterone secreted by MA-10 Leydig cells after a 4-h exposure to vehicle (DMSO, white bar), Fsk (10 M, black bar), or Fsk plus ryanodine (20, 40, 75, 150 M, gray bars). Results represent data obtained from three independent experiments, each performed in duplicate. C, Progesterone levels from MA-10 Leydig cells were measured after a 4-h exposure to vehicle (DMSO, white bar), Fsk (10 M, black bar), or Fsk plus dantrolene (Dtrl) (40, 80 M, gray bars). D, Progesterone levels from MA-10 Leydig cells were measured after treatments with vehicle (DMSO, white bar), Fsk (10 M, black bars), or Fsk plus ryanodine (75 M, gray bars) for 2, 4, and 8 h as indicated. For B, C and D, results represent data obtained from three independent experiments each performed in duplicate. P 0.05 was considered statistically significant using a one-way ANOVA test. Different lowercase letters indicate a statistically significant difference.

Article Snippet: Chemicals The RyR inhibitors ryanodine and dantrolene and the CaM inhibitor W7 hydroxychloride were obtained from Tocris Bioscience (Minneapolis, MN).

Techniques: Blocking Assay, Enzyme-linked Immunosorbent Assay

Journal: Endocrinology

Article Title: The calcium signaling pathway regulates leydig cell steroidogenesis through a transcriptional cascade involving the nuclear receptor NR4A1 and the steroidogenic acute regulatory protein.

doi: 10.1210/en.2012-1767

Figure Lengend Snippet: FIG. 2. Blocking internal Ca2 release reduces Star expression in MA- 10 cells. A, Progesterone levels were measured from MA-10 Leydig cells treated for 4 h with vehicle (DMSO, white bar), OH-Chol (40 M, black bar), or OH-Chol plus ryanodine (Rya) (75 M, gray bar). B, STAR protein levels were determined by Western blot using whole-cell extracts from MA-10 Leydig cells treated for 4 h with vehicle, Fsk (10 M), or Fsk plus Rya (75 M). A representative blot from three independent experiments is shown. The bands were quantified using ImageJ software and analyzed using Student’s t test: *, P 0.05 was considered statistically significant. C, Star mRNA levels were determined by quantitative RT-PCR using total RNA isolated from MA- 10 Leydig cells treated for 2.5 h with vehicle (white bar), Fsk (10 M, black bar), or Fsk plus Rya (75 M, gray bar). Values were normalized to Rpl19 level, and results are shown as fold induction over vehicle

Article Snippet: Chemicals The RyR inhibitors ryanodine and dantrolene and the CaM inhibitor W7 hydroxychloride were obtained from Tocris Bioscience (Minneapolis, MN).

Techniques: Blocking Assay, Expressing, Western Blot, Software, Quantitative RT-PCR, Isolation

Journal: Endocrinology

Article Title: The calcium signaling pathway regulates leydig cell steroidogenesis through a transcriptional cascade involving the nuclear receptor NR4A1 and the steroidogenic acute regulatory protein.

doi: 10.1210/en.2012-1767

Figure Lengend Snippet: FIG. 3. Ca2-induced Star transcription requires the NR4A1/5A1 element. A, To locate the Ca2-responsive element within the murine Star promoter, MA-10 cells were transiently transfected with a series of 5 deletion constructs (980, 195, 144, 120, 95, and 70 bp) of the murine Star promoter and cells were treated with vehicle (white bars), 8Br-cAMP (0.5 mM, black bars), or 8Br-cAMP plus dantrolene (150 M, gray bars) for 4 h. Results from four independent experiments each in duplicate are shown as fold induction over vehicle SD. *, P 0.05; **, P 0.02. B, MA-10 cells were transiently transfected with a 980-bp mouse Star reporter, either wild type or harboring a mutation in the NR4A1/5A1 element at 100 bp. Cells were then treated with vehicle (white bars), 8Br-cAMP (0.5 mM, black bars), or 8Br-cAMP plus ryanodine (75 M, gray bars) for 4 h. Results represent data obtained from three independent experiments each performed in duplicate. P 0.05 was considered statistically significant using a one-way ANOVA test. Different lowercase letters indicate a statistically significant difference.

Article Snippet: Chemicals The RyR inhibitors ryanodine and dantrolene and the CaM inhibitor W7 hydroxychloride were obtained from Tocris Bioscience (Minneapolis, MN).

Techniques: Transfection, Construct, Mutagenesis

Journal: Endocrinology

Article Title: The calcium signaling pathway regulates leydig cell steroidogenesis through a transcriptional cascade involving the nuclear receptor NR4A1 and the steroidogenic acute regulatory protein.

doi: 10.1210/en.2012-1767

Figure Lengend Snippet: FIG. 4. NR4A1 expression is modulated by changes in internal Ca2 level. A, NR4A1 and NR5A1 protein levels were determined by Western blot using nuclear extracts from MA-10 Leydig cells treated for 1 h with vehicle, Fsk (10 M), or Fsk plus ryanodine (Rya) (75 M). A representative blot from three independent experiments is shown. Detection of lamin B was used as loading control. The bands were quantified using ImageJ software and analyzed using Student’s t test. *, P 0.05 was considered statistically significant. B, Nr4a1 mRNA level was determined by quantitative RT-PCR using RNA isolated from MA-10 Leydig cells treated for 1 h with vehicle, Fsk (10 M), or Fsk plus Rya (75 M). Values were normalized to Rpl19 level, and results are shown as fold activation over vehicle SD. *, Statistically significant difference (P 0.05). C, Transient transfections of the rat Nr4a1 promoter (747 to 50 bp) reporter were performed in MA-10 Leydig cells treated with vehicle (white bar), cAMP (0.5 mM, black bar), or cAMP plus Rya (75 M, gray bar). Results are shown as fold induction over vehicle

Article Snippet: Chemicals The RyR inhibitors ryanodine and dantrolene and the CaM inhibitor W7 hydroxychloride were obtained from Tocris Bioscience (Minneapolis, MN).

Techniques: Expressing, Western Blot, Control, Software, Quantitative RT-PCR, Isolation, Activation Assay, Transfection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CXCL9 causes heterologous desensitization of CXCL12-mediated memory T lymphocyte activation.

doi: 10.4049/jimmunol.1101293

Figure Lengend Snippet: FIGURE 6. CXCL9-induced reduction in CXCL12-dependent chemotaxis is blocked by specific Rac and CXCR3 inhibitors. (A) Inhibitory effect of CXCL9 on CXCL12-mediated chemotaxis of Th1 cells was quantified, and the influence of inhibitors, which mainly block identified signaling molecules downstream of the CXCR3 receptor, was investigated Data are based on experiments also shown in (B)–(I). The following inhibitor concentrations were used: Piceatannol, 100 mM; PP2, 10 mM; LY294002, 20 mM; Ro 31-8220, 5 mM; Go¨6976, 2 mM; Edelfosine, 20 mM; Thapsigargin, 2.5 mM; NIBR2130, 810 mM; and NSC23766, 50 mM. (B–H) Th1 cells were exposed to graded concentrations of CXCL9 (10 nM) and CXCL12 (10 nM); additionally, one fraction of the cells was costimulated with CXCL9 (10 nM) placed in the upper and lower wells. Moreover, some of the cells were prestimulated (1 h) and costimulated with specified inhibitors. (I) Th1 cells were pretreated with the Rac-specific inhibitor NSC23766 (100 mM) for 1 h, after which they were prestimulated with CXCL9 (10 nM), immediately washed, and exposed to graded concentrations of CXCL9 (10 nM) and CXCL12 (10 nM). Data are mean 6 SD of n = 3 in triplicates. *p , 0.05, **p , 0.01, ***p , 0.001, two-way ANOVA and Bonferroni posttest. ns, Not significant.

Article Snippet: Edelfosine, PP2, and Piceatannol were purchased from Tocris Bioscience (Bristol, U.K.).

Techniques: Chemotaxis Assay, Blocking Assay