Figure 3

Determination of the cutoff NFI value for detection of PPI. DNA constructs of 52 pairs of flag-bait and myc-prey were randomly coupled and transfected into HEK293 cells. For negative controls, each myc-prey was cotransfected with pflag-CMV-2. Cell lysates were incubated on 6 anti-FLAG spotted slides, and PPI were detected using monoclonal anti-myc-cy3 (1:200). Following scanning, no obvious interaction signals were detectable, thus each were considered as non-interacting protein partners. Negative FI and NFI values were discarded, and the cutoff NFI value of 151.2 was calculated as the mean plus two times the standard error of the positive values (red column).