Profiling Protein Complexes by PLA with DTM Figure 4. DTM readout of PLA on fixed cells. In situ PLA (red) and immunofluorescence (green) detection of SMAD4 in a) cells transiently transfected with SMAD4 and b) wt cells having only endogenous levels of SMAD4. c) DTM readout of the ratios between SMAD4 from transfected cells and wt cells: transfected/wt (i), wt/transfected (a dye-swap experiment) (ii), transfected/transfected (iii), normalized against the ratio of beta- actin. doi:10.1371/journal.pone.0040405.g004 do not compete with the binding sites for the targeted protein interactions.In conclusion, the PLA-DTM provides a scalable approach for investigating an extended range of molecular features and potential biomarkers in different types of biological samples. We believe the potential of our method to assess a broad range of pairwise interactions can have important applications in numerous biological, medical and biotechnological contexts. Next generation sequencing offers greatly enhanced opportunities for using PLA to decode patterns of protein interaction in normal physiology and in pathological lesions. Materials and Methods Solid Phase PLA SP-PLA was performed as previously described [23] with the following modifications. After incubation with 1 nM PLA probes, 50 ml ligation mixture was added containing 10 mM Tris-HAc, 10 mM MgAc, 50 mM KAc, 0.5 U T4 DNA ligase (Fermentas), 0.1 mM of either one of the two connector oligonucleotides preincubated with a three-fold excess of its cassette oligonucleotide (Table S2) and 0.08 mM ATP (Fermentas). The reaction was carried out for 5 min at room temperature (RT). After ligation the reactions were washed twice and thereafter treated with 30 ml exonuclease mixture (50 mM KAc, 20 mM Tris-HAc pH 7.5, 3 mM MgAc, 1 mM DTT (Invitro- gen), 5 U lambda exonuclease (New England Biolabs), 10 U phi29 DNA polymerase (Fermentas) and 0.05 mg/ml BSA (New England Biolabs)) for 30 min at RT. After two washes 15 ml of two reactions to be compared were pooled for joint analysis. PCR was performed in a total volume of 50 ml containing 50 mM KAc, 20 mM Tris-HAc pH 7.5, 3 mM MgAc, 1 mM DTT, 0.2 mM dNTPs containing dUTP instead of dTTP (Fermentas), 0.1 U uracil-DNA glycosylase (UDG; Fermentas), 1.5 U platinum Taq polymerase (Invitrogen) and 100 nM of forward and reverse universal PCR primers (Table S2). Amplification was carried out by incubating at 95uC for 2 min, followed by 20 cycles of 95uC for 15 sec and 60uC for 1 min. In situ PLA In situ PLA was performed as previously described [9]. Briefly, cells were cultured on BD FalconTM 8-wells culture slides (BD NJ USA) and fixed with 3% paraformaldehyde (Sigma) in phosphate- buffered saline (PBS) for 30 min at RT and permeabilized by treatment with 0.05% Triton in PBS. Chambers were removed and the areas with cells were demarcated using a hydrophobic pen (DAKO Cytomation) to establish separated reaction wells on the slide. Slides were then blocked with in situ blocking buffer (20% human serum (Jackson ImmunoResearch), 100 mg/ml salmon sperm DNA (Invitrogen), 5 mM EDTA and 0.05% Tween-20 in PBS) for 2 h at RT, followed by washing for twice, 2 min in PBS, 0.05% Tween-20 at RT. The PLA probes (Table S3) were added in 40 ml in situ blocking buffer supplemented with 2.5 mM cystein (Sigma), followed by 5 min washing in 10 mM Tris-HCl pH 7.5, 0.05% Tween-20 and then 262 min in Tris-buffered saline (TBS), 0.05% Tween-20 at RT. PLA probes were ligated by adding 125 nM of each connector and backbone oligonucleotide (Table S2), 10 mM Tris-HAc (pH 7.5), 10 mM MgAc, 50 mM KAc, 0.05 U/ml T4 DNA ligase, 250 mM NaCl, 0.25 mg/ml BSA and 0.05% Tween-20 to the slides and incubating at 37uC. Ligation reactions were stopped after 30 min, followed by 262 min washes in TBS with 0.05% Tween-20 at RT. Transfer of Ligation Products from in situ Slides to PCR For DTM readout, ligation products were released from the slides by adding 1 U proteinase-K (Fermentas) in 40 ml 50 mM KAc, 3 mM MgAc, 20 mM Tris-HAc and 1 mM DTT PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e40405