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. 2016 Apr 28;7:11416. doi: 10.1038/ncomms11416

Figure 2. Virotrap experiments for binary PPI detection.

Figure 2

(a) Virotrap experiments for reciprocal detection of binary protein–protein interactions. HEK293T cells were transfected with GAG-bait constructs and E-tagged prey constructs. Additional co-transfection of VSV-G/FLAG-VSV-G expression constructs allowed efficient purification, which was followed by direct on-bead lysis and analysis by western blotting using anti-E-tag (for the presence of the prey protein), anti-GAG (bait expression levels and particles) and anti-β-actin antibodies. A representative experiment is shown for three biological repeat experiments. (b) Virotrap analysis of weak protein–protein interactions. Cells were transfected with MYD88 TIR bait constructs or control bait plasmids (GAG-EGFP) and combined with MYC-tagged MAL prey constructs or FADD prey controls. In the western blot analysis, proteins were revealed using anti-MYC for prey presence, anti-GAG for bait expression and VLP formation, and anti-β-actin for normalization of lysates. Analyses were performed for VLPs and for lysates of the producer cells. A representative experiment for three biological experiments is shown. Uncropped gel images and molecular weight markers are presented in Supplementary Fig. 10.