In Vitro Protein-Protein Binding Assay

An in vitro protein-protein binding assay was performed as previously described [14 (link)]. pGBKT7-VP1 bait vector and pGADT7-MAT1 prey vector were used as templates to transcribe and translate in vitro, then labeled with ³⁵S-Methionine (Amersham Pharmacia Biotech) in vitro transcription-translation system (Promega), respectively, to obtain ³⁵S-labeled fusion proteins HA-MAT1 and c-Myc-VP1. The ³⁵S-Methionine-labeled HA-MAT1 and c-Myc-VP1 were incubated at room temperature, then incubated with antibody against c-Myc (Clontech) in lysis buffer, subsequently mixed with protein A/G plus-agarose (Thermo Fisher, USA) and incubated for 3 h at 4°C. The beads were washed three times with lysis buffer. The radioactive antibody–protein complexes were eluted, and subjected to SDS/PAGE and then autoradiography.