Pegfp c1 er alpha

ERDDB was constructed by amplifying each domain using polymerase chain reaction (PCR), digestion, and ligation, according to the sequence shown in Fig. 2E. CyOFP1 and HaloTag were amplified from pKK-TEV-CyOFP1 (Addgene #105799) and pHAGE-EFS-MCP-HALOnls (Addgene #121937), respectively, and digested using BamHI and XhoI. The EV linker and P2A peptide sequences were amplified from pGEMT-TPE2A-Mef2c-Tdtomato-Gata4-Tbx5 (Addgene #111818) and pCAGGS-6011nes (Addgene #108652), respectively, and then digested with BspEI/MluI for the α/α and α/β ERDDB or BspEI/NotI for the β/β ERDDB. Nano-luciferase was amplified from pcDNA3.1-CAG-NanoLuc-VChR1-EYFP (Addgene #114112) and digested with NotI/XbaI. ERα and ERβ were amplified from pEGFP-C1-ER alpha (Addgene #28230) and pEGFP-C1-ER beta (Addgene #28237), respectively. For the α/α and α/β ERDDBs, the amplified construct was digested with XhoI/BspEI or XbaI/PspOMI, respectively, and for β/β ERDDB, with XhoI/BspEI or MluI/NotI. For lentiviral production, all ERDDBs were digested with BamHI/PspOMI and ligated into the pLenti-puro vector (Addgene #39481).

The ER(α) expression plasmid pEGFP-C1-ER alpha, ER(β) expression plasmid pCDNA3.1-nv5-ER beta and scramble vector Pbabe-neo were purchased from Addgene (Addgene plasmids #28,230, #22,770, and #1767, respectively). After being transformed using the heat shock technique, the Escherichia coli DH5α strain was spread using a sterile loop onto a prepared lysogeny broth (LB) agar plate containing kanamycin or ampicillin respectively, to isolate individual colonies of bacteria carrying the plasmids cited above and incubated overnight at 37 C. After 24 h, one colony was transferred into LB media with the corresponding antibiotic and incubated at 37 C for while shaking. After incubation, bacterial growth was characterized by a cloudy haze in the media. The plasmids were extracted and purified from the transformed and proliferated Escherichia coli DH5α using the GenElute HP Plasmid Maxiprep kit (Sigma‒Aldrich). MDA-MB-231 cells were then transfected using the traditional protocol with Attractene Transfection Reagent (Qiagen) following the manufacturer’s instructions. After 48 h of transfection, RNA and proteins were extracted as previously described.