Preceiver m02

After cloning and amplifying the UCP1 promoter region, the PCR products were cloned into the multiple cloning sites of pGL4.10. HEK-293T cells were co-transfected with pGL4.10-UCP1-prom- oter, PGL4.74 (Promega) and pReceiver-M02 (Genecopoeia) using Lipo3000 Transfection Reagent (Thermo Fisher Scientific, USA). After 48 h, cells were lysed, 100 μL of the cell lysate was added to each well, and the cells were incubated on a shaker at room temperature (24–26 °C) for 15 min. The cell lysate (20 μL) was added to 100 μL of luciferase assay reagent II (LARII) automatically by the machine. Next, 100 μL of Renilla luciferase assay reagent was added to the mixture to determine Renilla luciferase activity. The ratio of firefly luciferase and TK Renilla luciferase activity represented the reporter gene activity value. TK Renilla luminescence value was used as an internal reference. The experiment was performed in triplicate and expressed as the mean ± SD.

Plasmids carrying TRPV3 (pReceiver-M02), TRPV4 (pReceiver-M29), and TRPA1 (pReceiver-M29) were bought from Genecopoeia (Rockville, MD, USA). Using the Quick Change II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA), mutant TRPV3 channels were generated using the PCR-based site-directed mutagenesis method. The forward and reverse primers for the G573S mutant were 5′-GGGGTTTCCAGTCCATGAGCATGTACAGCGTCATG-3′ and 5′-CATGACGCTGTACATGCTCATGGACTGGAAACCCC-3′, respectively. The forward and reverse primers for the G573C mutant were 5′-GGGGTTTCCAGTCCATGTGCATGTACAGCGTCATG-3′ and 5′-CATGACGCTGTACATGCACATGGACTGGAAACCCC-3′, respectively. Sequencing was used to confirm mutagenesis.
TRPV3 (WT and mutants), TRPV4, and TRPA1 plasmids were transiently transfected into HEK 293T cells, as previously described [45 (link),46 (link)]. pEGFP (pEGFP-N1, Life Technologies) was co-transfected with the plasmid carrying TRPV3 WT or the TRPV3 mutants into HEK 293T cells to identify transfected cells. Turbofect reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used for transfection. All experiments were performed after a further 24–36 h incubation after transfection.