Fig. 3: Microscale thermophoresis confirms the absence of selectivity of BdLYR2 between LCO and CO.
a SDS-PAGE and coomassie blue staining of purified BdLYR2-ECR or BdLYR1-ECR and 400 ng or 200 ng of BSA respectively. b Schematic representation molecules used for MST: Unlabeled ligands and ECR labeled with a red fluorophore. c Curves of LCO and CO binding to BdLYR2-ECR. 20 nM of BdLYR2-ECR were incubated with a range of LCO-V(C18:1,NMe,S), CO4, CO5 or CO7. The plots show the fraction bound (mean ΔFnorm values divided by the curve amplitude), from 2 independent experiments. Plots showing the binding amplitudes (ΔFnorm) are in Supplementary Fig. 5. d Affinity of BdLYR2-ECR for LCO and CO, deduced from the binding curves. e Curves of LCO and CO binding to BdLYR1-ECR. 20 nM of BdLYR1-ECR were incubated with a range of LCO-V(C18:1,NMe,S), CO4, CO5 or CO7. Plots with binding amplitudes are shown in Supplementary Fig. 5. f Affinity of BdLYR1-ECR for LCO and CO, deduced from the binding curves. Note that since the curves of LCO binding are not saturated, the Kd might be overestimated. g Schematic representation molecules used for MST: fluorescent CO5-BODIPY and unlabeled ECR. h Curves of BdLYR1-ECR and BdLYR2-ECR binding to CO5. 100 nM of CO5-BODIPY were incubated with a range of BdLYR1-ECR or BdLYR2-ECR. Because CO5-BODIPY fluorescence changed upon binding to ECRs, fluorescence intensity rather than thermophoresis was analysed. The plots show the fraction bound (fluorescence intensity values divided by the curve amplitude), (f). Affinity of BdLYR1-ECR and BdLYR2-ECR for CO5, deduced from the binding curves. Note that since the curves for BdLYR1-ECR in (h) are not saturated, the Kd might be overestimated.
