Ibright analysis software version 5

To evaluate the correct molecular weight of the different fusion proteins, the supernatant of transfected HEK293T cells was run with β-mercaptoethanol (Sigma-Aldrich, #63689) on a 4-20% Mini-Protean® TGX Stain-Free™ Protein Gel (BioRad, #4568093). After transfer to PVDF membranes (ThermoFisher, #88518), the membranes were blocked with 1x Pierce™ Clear Milk Blocking Buffer (ThermoFisher, #37587) and incubated overnight with Mouse anti-Human IgG (CH3 domain) Secondary Antibody (Invitrogen, #MA5-16557). After washing with TBS-Tween (ThermoFisher, #28360), the membranes were incubated for 45 min with Anti-mouse IgG HRP-conjugated (R&D systems, #HAF007) and developed using SuperSignal™ West Atto Ultimate Sensitivity Substrate (ThermoFisher, #A38555). Images were obtained using the iBright FL1500 Imaging System (Invitrogen, #A44241) and analyzed with iBright Analysis Software Version 5.1.0 (Invitrogen).

To evaluate the correct molecular weight of the different fusion proteins, the supernatant of transfected HEK293T cells was run with β-mercaptoethanol (Sigma-Aldrich, #63689) on a 4-20% Mini-Protean® TGX Stain-Free™ Protein Gel (BioRad, #4568093). After transfer to PVDF membranes (ThermoFisher, #88518), the membranes were blocked with 1x Pierce™ Clear Milk Blocking Buffer (ThermoFisher, #37587) and incubated overnight with Mouse anti-Human IgG (CH3 domain) Secondary Antibody (Invitrogen, #MA5-16557). After washing with TBS-Tween (ThermoFisher, #28360), the membranes were incubated for 45 min with Anti-mouse IgG HRP-conjugated (R&D systems, #HAF007) and developed using SuperSignal™ West Atto Ultimate Sensitivity Substrate (ThermoFisher, #A38555). Images were obtained using the iBright FL1500 Imaging System (Invitrogen, #A44241) and analyzed with iBright Analysis Software Version 5.1.0 (Invitrogen).