Lanthascreen tr fret car coactivator binding assay

The LanthaScreen^® TR-FRET CAR Coactivator Binding Assay (Thermo Fisher Scientific, Waltham, MA, United States) was performed according to the manufacturer’s protocol with some modifications described in our previous paper (Carazo Fernandez et al., 2015 (link)). The LanthaScreen^® TR-FRET GR assay was performed following the protocol of the manufacturer. Results were obtained after fluorescence signal analysis with appropriate filters using the Synergy Biotek plate reader (BioTek Instruments Inc., Winooski, VT, United States). Results were calculated as relative fold interaction of a nuclear receptor with the coactivator fragment to the control (vehicle-treated, DMSO 0.1%) signal set to 1. Data are presented as the means and SD of three different experiments (n = 3) performed in quadruplicates.

The LanthaScreen^® TR-FRET CAR Coactivator Binding Assay (Thermo Fisher Scientific, Waltham, MA, USA) was used with some modifications to the manufacturers’ protocol reported in our previous paper [27 (link)]. In order to allow better interaction of benzodiazepines with the CAR LBD, incubation time was extended to 3 h. For fluorescence measurement, the Synergy Biotek plate reader was used with appropriate filters (BioTek Instruments Inc., Winooski, VT, USA). Results were calculated as an interaction of the CAR-LBD with the coactivator fragment relative to the control (DMSO 0.1%). Data are presented as the means and SD of a technical quadruplicate (n = 4).