Pcdna3 connexin43 gfp apex2

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.

To generate the Sig1R-GFP-APEX2 fusion construct, the DNA sequence between the EcoRI and BamHI digestion sites flanking the human Sig1R gene (SIGMAR1) was amplified by PCR from pCI-neo- Sig1R-3XFLAG. The DNA sequence between BamHI and NotI digestion sites flanking GFP-APEX2 was amplified from the plasmid pcDNA3 Connexin43-GFP-APEX2 (Addgene, cat#49385). The amplified Sig1R and GFP-APEX2 DNA fragments were then ligated into an empty vector of pEGFP-N1 (Clontech) cleaved by EcoRI and NotI. To generate the Sig1RN80-GFP-APEX2 construct for expressing a partial Sig1R molecule that includes amino acids 1-80 (Sig1RN80), we substituted the full-length Sig1R gene with the Sig1RN80 DNA fragment that was PCR-amplified. The Sig1R-GFP fusion construct was generated by inserting the full length Sig1R sequence into EcoRI and BamHI digested pEGFP-N1.