Duolink in situ proximity ligation assay detection kit

In situ protein-protein interactions were detected using the Duolink in Situ proximity ligation assay (PLA) detection kit (Sigma-Aldrich) following the manufacturer’s instructions. Cells were cultured in 8-well chamber slides at a low density and treated with acidic bile salts (100 μM, pH4) for 1h. After treatment, the cells were washed in PBS and fixed with 4% paraformaldehyde buffer for 45 min at RT. Cells were then permeabilized in 0.5% Triton X-100 in PBS for 5 min at RT, blocked for 45 min at RT with gentle shaking and incubated overnight at 4°C with the two primary antibodies raised against the two proteins of interest, each from a different host species. The following primary antibodies (APE1, Mouse Monoclonal, ThermoFisher Scientific; pSTAT3^Y705, Rabbit Monoclonal, Abcam; pEGFR ^Y1068 , Mouse Monoclonal, Cell Signaling) were used. Hybridizations, ligations, washings, and detection steps followed the supplier’s protocol. After final washes in buffer B, cells were mounted using the Duolink in situ mounting medium with DAPI, sealed with nail polish, and allowed to dry for 15 min at RT before imaging using a Zeiss Confocal Fluorescence Microscope (Zeiss LSM 880).