Figure 3

Quantification of protein expression from differentially tagged ORFs. (A) Sample set-up for indirect and sandwich ELISA assays used to quantify protein expression of individual subunits and protein-protein interactions, respectively. (B) Indirect ELISA data for a representative set of 8 constructs sharing VSV and E tags. For any two tags, 8 different architectures are possible. In the first four constructs VSV-Tag (in blue) is fused to the first ORF (Med7) and E-Tag (in red) to the second ORF (Med21). In the next four constructs, which are “reciprocal” architectures, VSV-Tag is fused to the second ORF (Med21) and E-Tag to the first (Med7). Constructs with tags in common are assayed in tandem, replicate experiments. Reported values are normalized for protein concentration and background. Control experiments include antibody drop-outs (not shown) and protein extracts lacking epitope tags. (C) ELISA values plotted into “heat maps” to better visualize patterns of expression. The top two maps show averaged values from replicate experiments, while the bottom two maps show the corresponding standard deviations. Maps on the left show Med7 expression patterns from the first ORF position (on the “y” axis) and should be read from left to right. Maps on the right show Med21expression patterns from the second ORF position (on the “x” axis) and should be read from top to bottom. Red and yellow colors indicate high and low relative expression levels, respectively.