to each well on the culture slide, followed by incubation at 37uC for 1 h. Ligation products were transferred to a reaction tube and the protease was inactivated at 95uC for 20 min. After inactiva- tion, 5 ml of ligation products from transfected cells and wildtype (wt) cells, respectively, were pooled pair-wise, mixed with 40 ml PCR mix (details above), and amplified by PCR for 7 cycles of 95uC for 15 sec and 60uC for 1 min. Ten ml of PCR products was used for microarray readout. Translation of Molecular Interactions into a Reporter Molecule Library Before microarray readout, the Taq polymerase in the amplification reactions was inactivated by adding 1 U protein- ase-K to 25 ml PCR product mix. Incubation at 37uC for 1 h was followed by denaturation of the protease at 95uC for 20 min. Next, 10 U each of the restriction enzymes MboI (Fermentas) and NlaIII (New England Biolabs) were added to the proteinase-K-treated PCR products along with 0.4 mg/ml BSA, followed by incubation at 37uC for 2 h. After restriction digestion, the uracil residues in the amplified and digested fragments were degraded by adding 1 U UDG to each reaction, incubating at 37uC for 30 min, and then heating to 95uC for 20 min. DTM Readout DTM readout was performed essentially as previously described [21]. Briefly, to each reaction chamber on the microarray (pre- heated to 55uC) 50 ml ligation mixture was added, comprising 10 ml pre-treated PCR product at a final buffer concentration of 20 mM Tris-HCl pH 8.3, 35 mM KCl, 7.2 mM MgCl2, 3 mM NAD, 0.1% Triton X100, 0.05% BSA and 5 U Ampligase (Epicentre). The ligation reactions were incubated at 55uC for 1 h. The individual wells were washed with 1 ml 16 TNT buffer (100 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween 20), and the array was dissembled to wash the whole glass slide with 0.16 SSC (15 mM NaCl, 1.5 mM Na-citrate). Next, each microarray chamber received 50 ml RCA mixture comprising 33 mM Tris- HAc pH 7.9, 10 U phi29 polymerase, 0.5 mg/ml BSA, 200 nM dNTP, 10 mM MgAc, 66 mM KAc, 1% Tween-20, and 1 mM DTT, and incubated at 37uC for 30 min. The silicon mask was removed and the array washed with 16TNT buffer and rinsed with 0.16 SSC. After reassembling the reaction chamber to the array, 10 nM of each detection oligonucleotide (Table S2) were hybridized to the RCA products in array hybridization buffer at 55uC for 1 h. The arrays were washed in 16 TNT buffer and then in 0.16 SSC at 46uC for 15 min. Finally, the arrays were dried by centrifugation and they were scanned by a GenePix 4000B microarray scanner and processed by GenePix Pro 6.0 software. The fluorescence intensities are mean values for individual microarray features. The ratios of Cy5 and Cy3 were mean ratios of fluorescence intensities of triplicate array features. The normalization values were calculated by: Normalized Cy5 Cy3 ~ Cy5(Target) Cy5(Control) = Cy3(Target) Cy3(Control) : Heatmaps were generated by R 2.10.1/BioConductor algo- rithms. Supporting Information Figure S1 Schematic illustration of PLA analysis with DTM readout. a) The PLA probes are incubated with the samples to be examined. b) The oligonucleotides on pairs of PLA Profiling Protein Complexes by PLA with DTM probes that have bound their targets in close proximity are ligated pairwise to give rise to reporter DNA strands. In the ligation step a sample barcode is introduced in the ligation product via a cassette connector oligonucleotide to allow for dual color comparisons of results for two samples in the same array spots. c) The ligation products are treated with exonucleases prior to d) pooling of pairs of samples to be compared against each other, followed by amplification of the ligation products with PCR. e) The PCR products are then treated with restriction enzymes and f) UDG, to generate single stranded reporter molecules with barcodes identifying the targeted proteins at both ends. g) The reporter molecules are hybridized to oligonucleotides complementary to pairs of protein tags, on a microarray, thereby allowing the reporter strands to be ligated into circles. h) The circularized reporter DNA molecules finally template RCA, primed by the oligonucleotides on the array, and the RCA products are detected by hybridization with Cy3 or Cy5 labeled detection oligonucle- otides, depending on which sample tag was introduced in the reporter DNA circle. (TIF) Figure S2 Optimization of the generation of reporter DNA strands. a) The assay background was significantly reduced by treating the ligation products with exonucleases and optimizing the number of PCR cycles. Two individual PLA reactions for detection of 10 pM VEGF were carried out with oligonucleotide system A1A2 and C1C2. After ligation, the ligation products were treated with a 50 ml exonuclease mix containing 0.1 U/ml lambda exonuclease and 0.2 U/ml phi29 polymerase, 0.01 mg/ml BSA in 16 special buffer (50 mM KAc, 3 mM MgAc, 20 mM Tris-HAc pH 7.5, 1 mM DTT) at 37uC for 30 minutes, followed by washing in 16 PBS, 0.05% Tween20. The treated products were pooled and amplified by PCR. Untreated ligation products were pooled and used as assay controls. The PCR-amplified products were diluted according to the results from analytical realtime-PCR quantification with specific primer pair combinations: AC and CC. Signals generated from primer combination AC are defined as background, while signals generated from primer combination CC are defined as signals. b) The effect of the Taq polymerase was investigated by addition of proteinase K at different points after PCR in comparison to reactions where no proteinase K was added. c) The buffer was optimized so that all enzymatic reactions could be performed in the same buffer. VEGF detection by PLA performing ligation and PCR amplification in the standard PCR buffer and the special buffer designed for the assay. d) The enzymatic efficiencies in the two separate buffers were investigated by treating PCR-products with the restriction enzymes NlaIII and MboI and UDG. (TIF) Figure S3 Comparison of signal from matched or partially mismatched reporter strands on DTM. The signal from the array spot with both tags complementary to the single stranded ligation product is significantly greater than for array features where only one of the tags is complementary. (TIF) Figure S4 Antibody validation pipeline. All antibodies used for detection of NFkB-family proteins were validated by western blot to investigate if they could bind a protein of the estimated size, Co-IP to confirm that they could bind the native protein while interacting with its known interaction partners, and SP-PLA to find the best combination of binders to be used for PLA. a) PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e40405