Gfp apex2

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.