Sa hrp

A two-step process for the detection of GPCR bound fusion proteins was optionally applied to increase sensitivity. Following stimulation with the appropriate CM as described above, cells were rinsed 3 times with HBSS before being incubated with 100 mM H₂O₂ and biotinyl-tyramide (from the Tyramide Signal Amplification kit, ThermoFisher, used as directed) or 500 µM biotin-phenol in the TSA amplification buffer from the same kit for 15 minutes at room temperature. The GPCR-bound peroxidase catalysed the oxidation of biotin-phenol generating a very short-lived biotinphenoxyl radical which covalently tagged proteins proximal to the peroxidase. Following the oxidation of biotin-phenol, cells were rinsed 3 times with HBSS. Then, the cells were incubated 30 minutes with a HBSS solution containing 1% BSA to block the non-specific binding sites before being incubated with a horse radish peroxidase conjugate of streptavidin (SA-HRP; Invitrogen) for 1 hour. Finally, the cells were again rinsed 3 times with HBSS before the detection of the covalently bound biotin using TrueBlue™. An alternate protocol was also applied: SA-HRP was replaced by SA-Qdots-705 (dilution 1:200, ThermoFisher) and photographed as described^{23 (link)}.

We used a microplate enzyme-linked immunosorbent assay (ELISA) for measuring protein-protein interactions [22 (link)], as described in [14 (link)]. Purified DvCad1-CR peptides were biotinylated using sulfo-NHS (N-hydroxysuccinimide)-photocleavable biotin (Pierce, Rockford, IL, USA), dialyzed into 200 mM NaCl, 20 mM Na₂CO₃ (pH 8.0), and stored in aliquots at 4 °C. Microtiter plates (high-binding, 96-well Immulon 2HB plates; Thermo Fisher Scientific, Inc., Waltham, MA) were coated with 1.0 µg Cry3Bb/well in 50 μL coating buffer (100 mM Na₂CO₃, pH 9.6), with or without the 1000-fold molar excess unlabeled DvCad1-CR peptides. In competition binding assays, 20 nM biotinylated DvCad1-CR peptide was mixed with increasing concentrations of the unlabeled DvCad1-CR peptides. Cry3Bb toxin-coated or peptide-coated plates were washed and incubated with horse radish peroxidase-conjugated streptavidin (SA-HRP; Pierce), and then incubated with an HRP chromogenic substrate (1-Step Ultra TMB-ELISA, Thermo Fisher Scientific, Inc., USA), after washing, to detect bound SA-HRP. Color development was stopped by adding 3M sulfuric acid, and absorbance was measured at 450 nm using a microplate reader (MDS Analytical Technologies, Sunnyvale, CA, USA). Data were plotted and analyzed using the Sigma Plot Version 11.0 (SPSS Science, Chicago, IL, USA).