TRPM8 N- and C-terminal tail GST-fusion proteins were produced and purified as described previously (Gkika et al., 2015 (link)). GST-fused purified proteins were then incubated with HEK cell lysates transfected with pEGFP-RAP-1, pEGFP-RAP-1N17, or pEGFP-RAP-1V12 plasmids (Bivona et al., 2004 (link)). For the direct interaction assay, the coding sequence of RAP-1-WT, RAP N17, and RAP V12 were subcloned in the pCMV TNT vectors (Promega) as EcoR1–Not1 fragments to produce them in vitro. Subsequently, Rap-WT and mutant proteins were translated in vitro using the TNT Quick Coupled Transcription/Translation Systems kit (Promega) and the FluoroTect GreenLys in vitro Translation Labeling System (Promega) as per the manufacturer’s instructions. For GST pull-down experiments performed with Rap1-WT loaded with GDP and GTP, in vitro translated Rap1-WT was incubated with 10 mM EDTA followed by 1 mM GDP or 100 μM GTPγS for 30 min at 30°C with constant agitation. The sample was then placed on ice, 60 mM MgCl2 was added, and the sample was vortexed.
Cell lysates or in vitro translated proteins were incubated overnight at 4°C together with the purified GST-fusion proteins. Subsequently, beads were washed extensively and bound proteins were eluted with SDS-PAGE loading buffer, separated on 4–20% wt/vol SDS-PAGE gels, and visualized by fluorescence imaging (Bio-Imager 600; GE Healthcare).
Cell lysates or in vitro translated proteins were incubated overnight at 4°C together with the purified GST-fusion proteins. Subsequently, beads were washed extensively and bound proteins were eluted with SDS-PAGE loading buffer, separated on 4–20% wt/vol SDS-PAGE gels, and visualized by fluorescence imaging (Bio-Imager 600; GE Healthcare).
