Pla assay

Cell culture and primary antibody staining were performed according to the classical immunofluorescence procedure. The PLA assay (Olink Bioscience) was conducted using the manufacturer's protocol. Dot quantification was achieved automatically using ImageJ software. Negative controls using only a single primary antibody are reported in Supplemental Figure S1D.

ESCs were fixed in 4% paraformaldehyde (PFA) for 20 min, washed, blocked and incubated with primary antibodies from different species following the protocol procedure. PLA assay (Olink Bioscience) was performed by incubating secondary antibodies conjugated with oligonucleotides (probes) for 1 h at 37 °C followed by hybridization and ligation with the PLA probes. Amplification of the occurred ligation was performed by in situ amplification in the presence of fluorescent-labelled oligonucleotides. Images were acquired using Leica TCS SP5 confocal microscope, with HCX PL APO × 40/1.25 objective. The signal intensity of the proximity reactions was quantified using Volocity Analysis software (PerkinElmer).