Engineered Connexin43 Constructs for Cellular Analysis

The ER-retention motif RRRRISLS was subcloned into a pENTR-V5-Cx43 vector to generate Cx43^RRRRISLS. Plasmids expressing ^GFPCx43, ^GFPmCherryCx43, Cx43^Avi and BirA-NLS were generated by cloning the appropriate cDNA into a pENTR vector containing the indicated tags. Plasmids expressing Cx43^ΔNLS were generated by amplifying cDNA encoding the first 236 and last 91 amino acids of rat Cx43, followed by a second PCR to unite the fragments; and finally cloned into a pENTR V5 vector. NLS-EGFP was kindly provided by Rob Parton (Addgene #67652) [69 (link)], mEmerald-Nesprin3-C-18 from Michael Davidson (Addgene #54203), Emerin pEGFP-N2 (588) from Eric Schirmer (Addgene #61985) [70 (link)], pDESTmycYBX1 from Thomas Tuschl (Addgene #19878) [71 (link)], pEGFP-C2 RanGAP from Mary Dasso (Addgene #13378) [72 (link)], pEGFP-N1 full length Importin- β was a gift from Patrizia Lavia (Addgene #106941) [73 (link)], pcDNA3 Connexin43-GFP-APEX2 (Addgene #49385) and pDisplay-BirA-ER (Addgene #20856) from Alice Ting [74 (link),75 (link)]. Experiments were performed 24 h after transient transfection of cells with Lipofectamine 2000 (Thermo Fisher Scientific), according to the manufacturer.

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Martins-Marques T., Witschas K., Ribeiro I., Zuzarte M., Catarino S., Ribeiro-Rodrigues T., Caramelo F., Aasen T., Carreira I.M., Goncalves L., Leybaert L, & Girao H. (2023). Cx43 can form functional channels at the nuclear envelope and modulate gene expression in cardiac cells. Open Biology, 13(11), 230258.

Publication 2023

Emerin pEGFP-N2 (588) pEGFP-N1 pcDNA3 Connexin43-GFP-APEX2 pDisplay-BirA-ER Lipofectamine 2000

Corresponding Organization : Escola Superior de Tecnologia da Saúde de Coimbra

Other organizations : Ghent University, Vall d'Hebron Institut de Recerca, Vall d'Hebron Hospital Universitari, Centro de Investigación Biomédica en Red de Cáncer, Instituto de Salud Carlos III

Top 5 similar protocols

Variable analysis

independent variables

Cx43RRRRISLS
GFPCx43
GFPmCherryCx43
Cx43Avi
BirA-NLS
Cx43ΔNLS

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

controls

Positive controls: NLS-EGFP, mEmerald-Nesprin3-C-18, Emerin pEGFP-N2 (588), pDESTmycYBX1, pEGFP-C2 RanGAP, pEGFP-N1 full length Importin-β, pcDNA3 Connexin43-GFP-APEX2, pDisplay-BirA-ER

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