Journal: PLoS ONE

Article Title: NCAM180 Regulates Ric8A Membrane Localization and Potentiates β-Adrenergic Response

doi: 10.1371/journal.pone.0032216

Figure Lengend Snippet: A- Ric8A-GFP co-transfected with NCAM120, NCAM140 or NCAM180 in HEK293T cells co-localizes specifically with the NCAM180 isoform. Scale bar: 10 µm. B- Ric8A-GFP or PmaxGFP plasmids were transfected in cortical neurons for 24 h. Images were taken focussing at the membrane plane with a 20× objective. NCAM staining is clearly associated to the cell membrane (B, E, K). Ric8A-GFP is strongly localized at the membrane at the level of the cell body and processes (A, D). PmaxGFP control plasmid is mostly found in the cytoplasm (J). Ric8A-GFP, but not PmaxGFP colocalizes with NCAM at the membrane (Ric8A/NCAM merge: C, F versus Pmax/NCAM merge: L). (G–I): higher magnification pictures (dotted line box D–F) using a 63× oil immersion objective indicate a strong co-localization of Ric8A-GFP with NCAM in the growth cone (I). Scale bars: 10 µm (A–F, J–O); 1 µm (G–I).

Article Snippet: Transfections of siRNA (0.1 nmol per 10 6 cells) were performed using the Amaxa electroporation system (kit V for HEK293T cells and mouse neuron nucleofector kit for neurons) as indicated by the manufacturer (Lonza).

Techniques: Transfection, Staining, Plasmid Preparation

Journal: PLoS ONE

Article Title: NCAM180 Regulates Ric8A Membrane Localization and Potentiates β-Adrenergic Response

doi: 10.1371/journal.pone.0032216

Figure Lengend Snippet: A- Representative experiment (out of 3 performed) of isoproterenol (10 µM) induced cAMP production in HEK293T cells transfected or not with NCAM180, in the presence (siCtrl) or absence (siRic8A) of Ric8A. Data are expressed as mean +/− SEM of 3 independent wells and analyzed by ANOVA and Bonferroni post-hoc test. B- Increase of cAMP production by Ric8A overexpression in HEK293T cells transfected for 48 h with Ric8A-GFP (▪) or PmaxGFP (⧫). Data are expressed as mean +/− SEM of 3 independent wells and analyzed by the t-test. C- HEK293T cells were transfected with NCAM180, and control or Ric8A siRNA for 24 h. Anti-NCAM beads were used for immunoprecipitation of cell extracts, A Western blot with anti-NCAM (first box) indicates that NCAM180 has been immunoprecipitated. A Western blot was also performed with anti-G αs on each extract before immunoprecipitation to check the amount of loaded G αs (second box). Anti-G αs (third box) and anti-Ric8A (fourth box) were used on NCAM180 immunoprecipitated extracts and show that Ric8A is significantly decreased by siRic8A compared to siCtrl, and that G αs is immunoprecipitated when Ric8A is present (siCtrl).. D- Amount of cAMP generated by isoproterenol (10 µM) that led to maximal efficacy in HEK293T, or HEK293T cells transfected with the different NCAM isoforms in the presence (siCtrl) or absence (siRic8A) of Ric8A. Data are expressed as % of siCtrl transfected cells and as mean +/− SEM of at least 3 independent experiments. Data were analyzed by ANOVA for more than 2 groups and Bonferroni post-hoc test. E- P-ERK1/2 induction by isoproterenol (10 µM) in hippocampal neurons from NCAM +/+ and −/− E15.5 embryos, in the presence (siCtrl) or absence (siRic8A) of Ric8A. Actin was used as constitutive protein. F- Amount of cAMP produced by NCAM +/+ and NCAM −/− non treated hippocampal neurons or after treatment with 1 µM isoproterenol in the presence (siCtrl) or absence (siRic8A) of Ric8A. Data are expressed as percentage of non-treated neurons (mean +/− SEM of 3 independent cultures). One-way ANOVA for more than 2 groups was performed. The effect of the single factor ‘isoproterenol treament’ was then analysed by the Bonferroni post-hoc test.

Article Snippet: Transfections of siRNA (0.1 nmol per 10 6 cells) were performed using the Amaxa electroporation system (kit V for HEK293T cells and mouse neuron nucleofector kit for neurons) as indicated by the manufacturer (Lonza).

Techniques: Transfection, Over Expression, Immunoprecipitation, Western Blot, Generated, Produced