Novablue singles competent cells

DNA for the BRCT domains of human BARD1 was fused with a GST tag and the construct subcloned into the bacterial expression vector, pETBlue-1 with NovaBlue Singles Competent Cells (Millipore-Sigma). The expression constructs were then transformed into Tuner (DE3)pLacI (Millipore-Sigma), a derivation of BL21 strain, for inducible overexpression. Fusion proteins were induced with a standard IPTG method for 3 h and crude bacterial lysate was prepared according to manufacturer’s protocol. The GST pulldown assay was performed with Pierce GST Protein Interaction Pull-Down Kit (ThermoFisher Scientific) using GST-BRCT and either WT or mutant 6His-p50 as indicated. GST fusion proteins were immobilized on glutathione-Sepharose beads and after washing the interacting complex was eluted and analyzed on SDS–PAGE followed by immunoblotting.

The WT rat TRPV1 (Caterina et al., 1997 (link)) and TRPV2 (Caterina et al., 1999 (link)) channel cDNA were provided by Dr. David Julius (UCSF, CA), and mouse TRPV3 (Peier et al., 2002 (link)) was provided by Dr. Feng Qin (SUNY Buffalo, NY). All constructs were cloned into modified pcDNA3.1(+) and pcDNA1 for high and low levels of expression, respectively. CBD binding site mutants were generated using the two-step PCR method using Phusion High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA), T4 ligase Quick Ligation kit (New England Biolabs) and NovaBlue Singles competent cells (MilliporeSigma, Burlington, MA) and Sanger-sequenced to check for PCR errors. Cumulative pore mutants were generated by Gibson assembly (GeneArt Gibson Assembly kit, Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s instructions and using a gBlock (Integrated DNA Technologies, Coralville, IA) encompassing nucleotides 1695–1993 in the rTRPV1 coding region.