Neutravidin resin

Cells expressing GFP-Hec1 were lysed and subjected for sonication in Lysis 125 buffer. Cell extract was first pre-clarified with neutravidin-resin (Thermo Fisher Scientific Inc., Waltham, MA), and then incubated with the neutravidin-resin conjugated with compound-biotin for 3 hrs at 4ºC. The resin was then collected, and washed with 50 resin volumes washing buffer (Lysis 125 buffer with 0.15% trion-x-100). Finally, the proteins on resin were eluted with 2x SDS loading buffer by boiling the resin for 10 min at 95°C heat plate. The supernatants were subjected to SDS-PAGE and Western blot analysis.

The composition of buffers, binding conditions and peptides sequences are described in Supplementary Methods. Peptides were tagged with Biotin at the N-terminus (Supplementary Table S1). For the resin-based binding assay, peptides (50 ng) were immobilized onto Neutravidin resin (Thermo-Scientific, Waltham, MA, USA). The immobilized peptides were incubated with purified Gβγ (Millipore, Billerica, CA, USA) (50 ng, 30 min, at 4 °C). Unbound Gβγ (flow-through from binding step) was collected and used as a loading control. Samples were analyzed by SDS-PAGE followed by western blotting (Gβ antibody (T-20, Santa Cruz, Dallas, TX, USA)). Densitometry analysis was performed with Imagelab5.0 (Bio-Rad, Hercules, CA, USA). For the plate-based in vitro binding assay, peptides were immobilized in neutravidin pre-coated 96-well plates (Thermo-Scientific). The immobilized peptides were incubated with purified Gβγ (Millipore) (30 min, RT). The quantification of bound Gβγ was determined by immuno-detection using the QuantaBlu kit (Thermo-Scientific), and measured with a plate reader (Tecan, Mannedorf, Switzerland, Exc.420/Emiss. 325 nm). Results are shown as relative binding compared to control group. The data are presented as mean ± s.e.m. Statistics were performed using a two-tail Student’s t-test with an accepted significance level at P<0.05.