Pcre luc

All the fragments were ligated into pcDNA3.1(+) plasmid, and the constructed plasmids were then verified by DNA sequencing. The pCre-luc (Santa Cruz, CA, USA) was kindly gifted by Xin Xie’s Lab of Tongji University. Human α-melanocyte stimulating hormone (α-MSH) and human adrenocorticotropin ACTH (1–24) was synthesized by Genescript (Nanjing, China). We purchased the paraformaldehyde (4%), phosphate buffered saline (PBS), bovine serum albumin, non-fat milk powder and β-mercaptoethanol from Sangon Biotech (Shanghai, China). TRNzol Universal Reagent and FastQuant RT Kit (with gDNase) were obtained from Tiangen Biotech (Beijing, China). Tetramethylbenzidine (TMB) chromogen solution was purchased from Beyotime^® Biotechnology (Shanghai, China). Hydrochloric acid and sulfuric acid were obtained from Sinopharm Chemical Reagent Co., Ltd (Beijing, China). The antibodies used in this study included Rabbit anti-Flag (Cell Signaling Technology, USA), Mouse anti-HA (Sigma Aldrich, MO, USA), Mouse anti-Flag (Abcam, Cambridge, UK), Goat anti-Mouse IgG (horseradish peroxidase (HRP)-conjugated) (ABclonal Biotech Co., Ltd, Wuhan, China), and Goat Anti-Rabbit Alexa-Fluor 594 (Abcam). All primers used for full-length gene amplification, reverse transcription PCR (RT-PCR), and real-time quantitative PCR are listed in Supplemental Table 1 and synthesized by GENEWIZ (Suzhou, China).

Wild-type MRAP2 and MC4R were amplified from complementary DNA (cDNA) library of mouse brain tissue. The original protein sequence of the variants was obtained by PCR cloning from wtMRAP2, and the inverted sequence was synthesized from GENEWIZ (South Plainfield, NJ, USA). Finally, primers were designed to assemble the entire variant sequences. All plasmids were verified by DNA sequencing. 2FLAG-tagged/3HA-tagged wtMRAP2 and MRAP2 mutants, 3HA-tagged MC4R, 3HA-tagged D2dr, 2FLAG-tagged RAMP3, VF1-MRAP2, FLAG-MRAP2-VF2, and FLAG-MC4R-VF2 expression constructs were subcloned into pcDNA3.1 vectors. The pCre-luc (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and pRL-TK (Promega, Madison, WI, USA) plasmids were kind gifts from Xin Xie’s Lab of Tongji University. α-MSH was obtained from Genescript (Nanjing, China). 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution was purchased from Beyotime® Biotechnology (Shanghai, China). Mouse monoclonal anti-HA (Sigma-Aldrich, MO, USA), anti-FLAG (ABclonal Biotech Co., Ltd, Wuhan, China), and horseradish peroxidase (HRP)-conjugated antibodies against mouse (ABclonal Biotech Co., Ltd, Wuhan, China) were used in this study. The primers used in this study were all synthesized from GENEWIZ.