Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2020 Jul 15;14:214. doi: 10.3389/fncel.2020.00214

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

Copyright © 2020 Papon, Le Feuvre, Barreda-Gómez, Favereaux, Farrugia, Bouali-Benazzouz, Nagy, Rodríguez-Puertas and Landry.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

PMC Copyright notice

Fibulin-2 interacts with GABAB and alters antagonist-induced effects in vitro. (A) Fibulin-2-Flag co-immunoprecipitates with myc-GABAB1a (lane 1, left panel) in COS-7 cells. In the absence of HA-GABAB2, the co-immunoprecipitation is largely reduced (lane 2, left panel). Fibulin-2-Flag does not co-immunoprecipitate with the GABAB1b subunit (lane 3, left panel). No signal is observed in the absence of myc-GABAB1a or fibulin-2-Flag (lane 3 and 4, left panel). Conversely, myc-GABAB1a co-immunoprecipitates with fibulin-2-Flag (right panel). IP, immunoprecipitation; WB, Western-Blot. (B) GABAB1a-GFP subunit co-immunoprecipitates with HA-GABAB2 in COS-7 cells in the presence (lane 1, left panel) or in the absence (lane 2, left panel) of fibulin-2-Flag. Fibulin-2-flag transfection actually results in the expression of the protein (right panel). IP: immunoprecipitation; WB: Western-Blot. (C) One site fitting of the [³H]-CGP54626 binding sites (GABAB receptor) inhibited by baclofen in rat primary cell cultures (expressed in % of inhibition). The membrane preparations from cell cultures were incubated with 2 nM [³H]-CGP54626 and increasing concentrations of baclofen. The affinity of the GABAB receptor to the specific agonist baclofen was similar with fibulin-2 (); anti-fibulin-2 siRNA () controls (■). pIC50_control: –5.0 ± 0.1; pIC50_siRNA: –4.6 ± 0.1; pIC50_fibulin: –4.7 ± 0.1. (D) Effects of saclofen on FRET efficiency between GFP-GABAB1a and DsRed-GABAB2 ± baclofen at 10^–6M. Baclofen effect is reversed by saclofen at 100 μM and 1 mM. Application of anti-fibulin-2 siRNA (20 or 40 pmol) suppresses this reversion at 100 μM dose (***p < 0.001 “B1a/B2 + saclo100 μM” vs. “B1a/B2 + saclo100 μM + siRNA40”). At high dose (1 mM), saclofen remains efficient even in the presence of anti-fibulin-2 siRNA (NS: p > 0.05 “B1a/B2 + saclo1 mM” vs. “B1a/B2 + saclo1 mM + siRNA40”) (n = 21 cells). *p < 0.05; **p < 0.01; ***p < 0.001; n.s.: p > 0.05 vs. “B1a/B2 + baclofen” unless indicated. (E) Effects of CGP55845 on FRET efficiency between GFP-GABAB1a and DsRed-GABAB2 ± baclofen at 10^–6M. Baclofen effect is reversed by CGP55845 at 0.5 and 50 μM. Application of anti-fibulin-2 siRNA (40 pmol) suppresses this reversion at 0.5 μM dose (*p < 0.05 “B1a/B2 + CGP 0.5 μM” vs. “B1a/B2 + CGP 0.5 μM + siRNA40”). At high dose (50 μM), CGP remains efficient even in the presence of anti-fibulin-2 siRNA (NS: p > 0.05 “B1a/B2 + CGP50 μM” vs. “B1a/B2 + CGP50 μM + siRNA40”) (n = 21 cells). *p < 0.05; **p < 0.01; ***p < 0.001; n.s.: p > 0.05 vs. “B1a/B2 + baclofen” unless indicated.

Inline graphic — Fibulin-2 interacts with GABAB and alters antagonist-induced effects in vitro. (A) Fibulin-2-Flag co-immunoprecipitates with myc-GABAB1a (lane 1, left panel) in COS-7 cells. In the absence of HA-GABAB2, the co-immunoprecipitation is largely reduced (lane 2, left panel). Fibulin-2-Flag does not co-immunoprecipitate with the GABAB1b subunit (lane 3, left panel). No signal is observed in the absence of myc-GABAB1a or fibulin-2-Flag (lane 3 and 4, left panel). Conversely, myc-GABAB1a co-immunoprecipitates with fibulin-2-Flag (right panel). IP, immunoprecipitation; WB, Western-Blot. (B) GABAB1a-GFP subunit co-immunoprecipitates with HA-GABAB2 in COS-7 cells in the presence (lane 1, left panel) or in the absence (lane 2, left panel) of fibulin-2-Flag. Fibulin-2-flag transfection actually results in the expression of the protein (right panel). IP: immunoprecipitation; WB: Western-Blot. (C) One site fitting of the [³H]-CGP54626 binding sites (GABAB receptor) inhibited by baclofen in rat primary cell cultures (expressed in % of inhibition). The membrane preparations from cell cultures were incubated with 2 nM [³H]-CGP54626 and increasing concentrations of baclofen. The affinity of the GABAB receptor to the specific agonist baclofen was similar with fibulin-2 (); anti-fibulin-2 siRNA () controls (■). pIC50_control: –5.0 ± 0.1; pIC50_siRNA: –4.6 ± 0.1; pIC50_fibulin: –4.7 ± 0.1. (D) Effects of saclofen on FRET efficiency between GFP-GABAB1a and DsRed-GABAB2 ± baclofen at 10^–6M. Baclofen effect is reversed by saclofen at 100 μM and 1 mM. Application of anti-fibulin-2 siRNA (20 or 40 pmol) suppresses this reversion at 100 μM dose (***p < 0.001 “B1a/B2 + saclo100 μM” vs. “B1a/B2 + saclo100 μM + siRNA40”). At high dose (1 mM), saclofen remains efficient even in the presence of anti-fibulin-2 siRNA (NS: p > 0.05 “B1a/B2 + saclo1 mM” vs. “B1a/B2 + saclo1 mM + siRNA40”) (n = 21 cells). *p < 0.05; **p < 0.01; ***p < 0.001; n.s.: p > 0.05 vs. “B1a/B2 + baclofen” unless indicated. (E) Effects of CGP55845 on FRET efficiency between GFP-GABAB1a and DsRed-GABAB2 ± baclofen at 10^–6M. Baclofen effect is reversed by CGP55845 at 0.5 and 50 μM. Application of anti-fibulin-2 siRNA (40 pmol) suppresses this reversion at 0.5 μM dose (*p < 0.05 “B1a/B2 + CGP 0.5 μM” vs. “B1a/B2 + CGP 0.5 μM + siRNA40”). At high dose (50 μM), CGP remains efficient even in the presence of anti-fibulin-2 siRNA (NS: p > 0.05 “B1a/B2 + CGP50 μM” vs. “B1a/B2 + CGP50 μM + siRNA40”) (n = 21 cells). *p < 0.05; **p < 0.01; ***p < 0.001; n.s.: p > 0.05 vs. “B1a/B2 + baclofen” unless indicated.