Co-immunoprecipitation for Western blot analysis

For in vitro co-immunoprecipitation analysis, approximately 10% of the total recombinant proteins were used as input. To pull down the interacting proteins from the viruliferous planthoppers, total proteins were extracted from 4th-instar viruliferous planthoppers using 1× PBS buffer (pH 7.2) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific). Approximately 10% of the total protein fraction was reserved as input. Then, 10 μL of protein G beads (Thermo Fisher Scientific) was mixed with 2 μg of an anti-His/-Flag monoclonal antibody (Merck Millpore, Billerica, MA, USA) or anti-YY1 polyclonal antibody (Thermo Fisher Scientific) or a homemade anti-NP monoclonal antibody before being incubated with 200 μL of His- or Flag-tagged recombinant proteins for 15 min at room temperature. The expression products from the pET28a vector or the IgG antibody (Merck Millipore) were used as negative controls. Then, 500 μL of recombinant Flag- or His-tagged target proteins or the total proteins from viruliferous planthoppers were added and incubated at 4 °C overnight. Finally, the antibody-protein-protein complex was dissociated from the beads with the elution buffer (Thermo Fisher Scientific) for Western blot analysis.