Hrp conjugated goat anti rabbit antibody

After SDS-PAGE of the collected SEC fractions (10 μL per fraction) on a 4–15% acrylamide gradient gel, the proteins were transferred onto a PVDF membrane using a Trans-Blot Turbo semi-dry transfer system (Bio-Rad). In the cases where the cargo proteins were fusion constructs with N- or C-terminal parts of TccC3HVR, a custom-made anti-TccC3HVR rabbit polyclonal antibody (Cambridge Research Biochemicals) was used as the primary antibody at 1:1000 dilution. For Cdc42 without a TccC3HVR fusion, an anti-Cdc42 rabbit polyclonal antibody (Cell Signaling Technology, Cat. No. 2462) was used as the primary antibody at 1:1000 dilution. For TEV, an anti-TEV protease rabbit polyclonal antibody (Novus Biologicals, Cat. No. NBP1–97669) was used as the primary antibody at 1:500 dilution. An HRP-conjugated goat anti-rabbit antibody (Bio-Rad, Cat. No. 170–6515) was applied as the secondary antibody at 1:2000 dilution in all cases. Detection was performed with Western Lightning Plus ECL reagent (PerkinElmer, Cat. No. NEL104001EA) and imaged in a ChemiDoc MP imaging system (Bio-Rad).

Transiently transfected HEK293T cells were seeded in a poly-L-lysine coated 96-wells plate for 48 h at 37 °C and 5% CO₂. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% NP-40 as described above. In the case of determination of surface receptor expression levels, cells were only fixed with 4% paraformaldehyde. Receptor expression was detected using a Rat anti-HA antibody (1:1000 in 1% (v/v) FBS/PBS, Clone 3F10, Roche) and HRP-conjugated Goat-anti-Rat antibody (1:1000 in 1% (v/v) FBS/PBS, Pierce). Detection of intrabody-FLAG expression was done using a Mouse-anti-FLAG antibody (1:1000 in 1% (v/v) FBS/PBS, Clone M2, Sigma-Aldrich) and HRP-conjugated Goat-anti-Mouse antibody (1:2000 in 1% (v/v) FBS/PBS, Pierce). In the case of U251-iUS28 cells, receptor expression was detected using an Rabbit-anti-US28 antibody (1:1000 in 1% (v/v) FBS/PBS, Covance^{24 (link)}) and HRP-conjugated Goat-anti-Rabbit antibody (1:1000 in 1% (v/v) FBS/PBS, Bio-Rad). Incubation with antibodies was done for 1 h at RT. Wells were washed three times with 1× PBS in between all incubation steps. Antibody binding was detected using 1-Step ultra TMB-ELISA substrate (Thermo Fisher Scientific) and the reaction was stopped with 1 M H₂SO₄. Optical density was measured at 450 nm with a PowerWave plate reader (BioTek). Data were analyzed using GraphPad Prism version 8.0.