Figure 2. Analysis of the concentration range over which differences of protein concentrations can be recorded. a) 10- fold differences in concentration of VEGF between two samples were successfully detected even when the concentrations were as low as 100 and 10 fM. For normalization purposes IL8 was spiked into all samples at a fixed concentration of 10 pM, and the fluorescence ratio for VEGF was normalized against the fluorescence ratio for IL8. b) Two-fold differences of IL8 concentrations were readily detected. Plotted are the Profiling Protein Complexes by PLA with DTM fluorescence ratios when comparing a sample with the indicated concentration of IL8 and a sample with 10 pM IL8, after normalization for the fluorescence signal from detection of 10 pM VEGF. c) Detection of 10 pM VEGF over background in cell lysates. Two samples – cell lysate with or without VEGF spiked in – were compared. The results are plotted in an MA-plot with the log2 ratios between Cy5 and Cy3 (M = log2(Cy5/Cy3)) on the y-axis and the log2 average intensity of Cy5 and Cy3 (A = 0.5 log2(Cy56Cy3)) on the x-axis. Red, filled circles show triplicate measurements of VEGF. Blue, filled circles represents triplicate measures of a spiked in oligonucleotide control. The remaining unfilled circles represent the measurements of the other four targeted proteins (p50, RelA, RelB, IkBa) and all their possible pairwise interactions. Dotted lines indicate the 95% confidence interval (2 standard deviations from the mean ratio between Cy5 and Cy3). doi:10.1371/journal.pone.0040405.g002 antibodies was also evaluated to confirm detection of the proper target molecules. Moreover, we only employed antibodies that generated the same low signals in cell lysates whether the cells had been transfected with any other NFkB family member or not (data not shown). The targeted proteins of the NFkB family were transiently overexpressed and we analyzed differences in levels of proteins and of interacting proteins between two samples where either RelA or RelB was omitted from the transfections. Our results demonstrate that we can detect a difference in the abundance of the two proteins, and we could also demonstrate the relative levels of interactions (Figure 3, and Table S1a). In the experiment we used lysates from cells that had been transiently transfected with constructs encoding the NFkB proteins. We compared two lysates, one where the cells had been transiently transfected with RelA, p50 and IkBa and another transfected with the same proteins but replacing RelA with RelB. As expected we detected a greater abundance of RelB and of RelB in its known interaction with p50 in samples transfected with RelB as compared to samples transfected with RelA. Conversely, in samples transfected with RelA we saw a greater abundance of RelA and RelA interacting with its known partners p50 and IkBa as compared to the sample transfected with RelB. Similar results were observed when the experiment was replicated using the opposite sample barcodes for the two samples (Figure S5). To demonstrate the possibility to use microarray for analysis of PLA reactions performed in situ we designed an oligonucleotide system for readout either by DTM or in situ PLA with RCA. The oligonucleotides conjugated to antibodies were constructed to template ligation of two separate connector oligonucleotides to form a DNA circle when pairs of PLA probes co-localize by binding the same protein or two interacting proteins. One of the connector oligonucleotides comprises two PCR primer sites, and the other encodes the two tags for correct localization on the oligonucleotide arrays, and one of two possible sample tags used for fluorescent detection of RCA products generated in the array features. To demonstrate the performance of the method, we investigated levels of the SMAD4 protein in transfected and untransfected cells (Figure 4, experimental design described in greater detail in Figure S6). After ligation with different connector oligonucleotides for the two samples, the DNA circles were detected either by in situ PLA with RCA in cells prepared for microscopy, or by PCR amplification and then DTM readout. For in situ PLA detection (Figure 4a, b) phi 29 polymerase was then added to initiate RCA, followed by decoration of the amplification products by hybridization with fluorescent oligonucleotides. Relatively high endogenous SMAD4 expression was detected in the cells, while as illustrated by the single transfected cell in Figure 4a, only a minor proportion of the cells expressed the transgene. PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e40405