Immunoprecipitation with antibodies against p50, RelA and RelB showed that the known interaction partners were co-immunopre- cipitated together with the targeted protein. b) Different antibodies were used for capture (c), PLA probe 1 (p1) and probe 2 (p2) in SP-PLA. The combination with A301–823A as capture and one of the probes together with ab7970 as the other probe was superior in generating a high signal over background for detection of RelA when compared to using A301–823A as capture and ab7970 as the two PLA probes. (TIF) Figure S5 Validation of detection of protein-interaction between the NFkB-family proteins. The detection of protein interactions among the NFkB-family proteins was repeated with the two sample tag sequences, introduced during the ligation reaction, switched for the two samples. In this heat map negative log2 Cy5/Cy3 ratios (green) indicate a greater abundance in sample I (transfected with p50, RelA and IkBa), while positive log2 Cy5/Cy3 ratios (red) indicate a greater abundance in sample II (transfected with p50, RelB and IkBa). Raw data are presented in Table S1b. (TIF) Figure S6 Experimental setup for in situ PLA detection of Smad4 with fluorescent in situ detection and DTM readout. PLA probes directed to Smad4 and beta-actin were applied to slides with fixed cells that had or had not been transfected with Smad4. Connector oligonucleotides were added and joined into circular reporter molecules by enzymatic ligation. The reporter DNA circles were detected in situ in cells fixed on slides by allowing them to guide rolling circle amplification to generate large concatemers of DNA. The amplification products were then detected by hybridization with a fluorescence labeled detection oligonucleotide. For an alternative readout on micro- arrays, reporter DNA circles were released from slides by References 1. Vidal M, Cusick ME, Baraba´si A-L (2011) Interactome networks and human disease. Cell 144: 986–998. doi:10.1016/j.cell.2011.02.016. 2. Fo¨rster T (1948) Zwischenmolekulare Energiewanderung und Fluoreszenz. Annalen der Physik 437: 55–75. doi:10.1002/andp.19484370105. 3. Stryer L, Haugland RP (1967) Energy transfer: a spectroscopic ruler. Proc Natl Acad Sci U S A 58: 719–726. 4. Kenworthy AK (2001) Imaging Protein-Protein Interactions Using Fluorescence Resonance Energy Transfer Microscopy. Methods 24: 289–296. doi:06/ meth.2001.1189. 5. Shi Y, Huang W, Tan Y, Jin X, Dua R, et al. (2009) A Novel Proximity Assay for the Detection of Proteins and Protein Complexes: Quantitation of HER1 and HER2 Total Protein Expression and Homodimerization in Formalin-fixed, Paraffin-Embedded Cell Lines and Breast Cancer Tissue. 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(TIF) Table S2 List of oligonucleotides used in the experi- ments. Listed are the sequences of all oligonucleotides used in the experiments together with their modifications in the 39-free and 59-free ends respectively. (TIF) Table S3 List of antibodies used in the experiments. Listed are the antibodies with their target proteins, catalog number and manufacturer, any modification, type of immunoglobulin (Ig), and concentrations in various assays. (TIF) Supplementary Results and Materials and File S1 methods. (PDF) Acknowledgments We gratefully acknowledge that Dr. George Mosialos (Al. Fleming BSRC, Athens, Greece) kindly provided the p50 plasmid, and Dr. Mathias Howell offered valuable suggestions during the initiation of the project. Author Contributions Conceived and designed the experiments: MH RYN OE KP UL. Performed the experiments: MH RYN. Analyzed the data: MH RYN. Wrote the paper: MH RYN UL. 12. 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