cDNAs coding for full-length RARα, RXRα (referred to as RAR and RXR in this article) were purchased from the cDNA Resource Center, subcloned after PCR amplification into modified pEGFP-C3 (Clontech Laboratories) and pmCherry-C3 vectors using XhoI and HindIII sites. RAR and RXR ligand-binding domain sequences were acquired from UniProt (47 ). NR LBD (ligand-binding domain, lacking DBD) sequences were amplified using PCR, then subcloned into pEGFP-C3 (Clontech Laboratories) and pmCherry-C3 vectors using the same technique as that used for the full-length receptors. The EGFP-VDR construct was made in our department as described in a previous paper (21 ).
pLenti-C-Myc-DDK-IRES-Neo, a third-generation transfer vector (Origene) was digested with EcoRI and PmeI and subcloned with HaloTag, EGFP, and RARα sequence. Then it was modified by cutting out the HaloTag to suit cloning the NRs and the fluorescent proteins used in our laboratory. To clone mScarlet3-RXR, the newly designed pLenti-C-Myc-DDK-EGFP-RARα transfer vector was used. By using BsTB1 and Mlu1, EGFP was replaced by mScarlet3, then RXRα was inserted between MluI and Pmel using proper primers.
pLenti-C-Myc-DDK-IRES-Neo, a third-generation transfer vector (Origene) was digested with EcoRI and PmeI and subcloned with HaloTag, EGFP, and RARα sequence. Then it was modified by cutting out the HaloTag to suit cloning the NRs and the fluorescent proteins used in our laboratory. To clone mScarlet3-RXR, the newly designed pLenti-C-Myc-DDK-EGFP-RARα transfer vector was used. By using BsTB1 and Mlu1, EGFP was replaced by mScarlet3, then RXRα was inserted between MluI and Pmel using proper primers.