Pierce cell surface protein biotinylation and isolation kit

For the cell surface proteins isolation, Pierce™ Cell Surface Protein Biotinylation and Isolation Kit (Thermo Fisher Scientific) were used. The application and sample acquisition was performed according to the manufacturer’s instructions. Briefly, Sulfo-NHS-SS-Biotin solution was applied on cell cultures for 30 min at 4°C to ensure biotin binding. Afterward, the cells were collected and lysed. The cell lysate was then mixed with NeutrAvidin™ Agarose on a separation column and incubated for 30 min (at RT) with end-over-end mixing on a rotator to ensure the binding of NeutrAvidin™ to Sulfo-NHS-SS-Biotin-labeled proteins. The column was washed, and the flow-through containing the unlabeled (intracellular) proteins was captured to be used on Western blot (labeled as IC). The column was washed thoroughly (using 500 μL of washing buffer applied on the column, centrifuged to pass the buffer and repeated 3-times), and the Sulfo-NHS-SS-Biotin-labeled proteins were eluted from NeutrAvidin™ via treatment with a reducing agent (250 mM DTT). The eluted proteins (membrane-bound) were collected and used for Western blot detection (labeled as M).

Protein biotinylation was performed using the Pierce Cell Surface Protein Biotinylation and Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol. Cells labeled with sulfo-NHS-SS-biotin were subjected to pulldown using streptavidin-agarose beads for 3 h at 4 °C. The beads were re-suspended in 5× sample loading buffer and heated at 100 °C for 10 min prior to SDS-PAGE.

Proteins were biotinylated using the Pierce™ Cell Surface Protein Biotinylation and Isolation Kit (Thermo Fisher, MA, United States). Various concentrations of biotinylated Sip (NHS-Biotin-Sip) were added to BBMV in a range 25–100 nM at intervals of 25 nM. Additionally, 100 nM biotinylated Sip was separately added with a 10-, 15-, and 20-fold excess of unmarked Cry8Ca into the BBMV binding system. For the competitive binding assays, 0.1% BSA in PBS was added to all binding systems to make them up to 100 μl. All the systems were incubated for 1 h at room temperature. Unbound toxin was removed by centrifugation (10 min at 18,000 rpm), and the pellets were washed twice with PBS (pH 7.6, 0.1% BSA). The precipitate was suspended in 10 μl 1× SDS loading buffer, loaded on an SDS-PAGE gel, and electrotransferred to a PVDF membrane (GE Healthcare, MA, United States). The PVDF membrane was then incubated with Streptavidin-HRP (1:3000 dilutions) for 1 h at room temperature, followed by three wash cycles with PBST, 15 min per cycle. Binding was visualized using an ECL chemiluminescent kit (Thermo Scientific, MA, Untied States).

For isolation of membrane proteins, the Pierce Cell Surface Protein Biotinylation and Isolation Kit (ThermoFisher) was used according to the manufacturer's protocol. In brief, cells were cultured for 48 h and transfected with either the WT or one of the mutant plasmids. Cells were biotinylated, lysed and isolated by binding to probed agarose beads 24 h after transfection. After elution, the proteins were prepared for Western blot analysis. Western blots were performed and analysed as described above. A monoclonal mouse anti-actin primary antibody (1:30,000, A5441, Sigma-Aldrich) was used as control.

Proteins expressed on the cell surface were biotin-labeled with the Pierce Cell Surface Protein Biotinylation and Isolation Kit (ThermoFisher Scientific) as specified in the Supplemental Methods.