Ab75766

The in-situ proximity ligation assay (PLA) procedure was performed with the Duolink® PLA Kit (Sigma-Aldrich, DUO92008) and following the manufacturers protocol. The cells were incubated with the primary antibodies i.e., anti-PGRMC1 (Abcam, ab48012) with PHB1 (Abcam, ab75766) and PHB2 (Cell signaling, 14085S) overnight at 4 °C. The slides were washed twice for 5 min with buffer A, followed by incubation with the PLA probes (anti-goat PLUS and anti-rabbit MINUS) in antibody diluent for 60 min at 37 °C. After washing twice for 5 min with buffer A, ligation was performed using ligase diluted in ligation buffer for 30 min at 37 °C. Then the cells were washed with buffer A before incubation for 100 min with amplification stock solution at 37 °C. After washing twice for 10 min with buffer B, nuclear DNA was labeled with DAPI for 10 min and slides were mounted with mounting medium. Negative PLA control was performed using respective isotype control antibodies (isotype goat, Abcam, ab37373; isotype rabbit, Abcam, ab37415). Red fluorescence dots inside the cellular areas representing a single protein–protein interaction were quantified using image J software.

The proximity ligation assay (PLA) procedure was performed using the Duolink^® PLA Kit (Sigma-Aldrich, DUO92008) according to the manufacturer’s instructions. Cells were spun on glass slides, fixed and permeabilized as described above. Incubation with the primary antibody cocktail containing anti-PGRMC1 antibody (Abcam, ab48012) or anti-ERα antibody (Abcam, ab259427) with PHB1 (Abcam, ab75766) or PHB2 (Cell signaling, 14085S) antibody was performed overnight at 4 °C. Negative control PLA was performed using respective isotype control antibodies (goat isotype, Abcam ab37373; mouse isotype, Abcam ab37355; rabbit isotype, Abcam ab37415). Nuclear DNA was labeled with DAPI for 10 min and slides examined by fluorescence microscopy within one week after storage at 4 °C in the dark. Each red dot represented a single interaction. Dots per cell were quantified using imageJ software [38 (link)].

Cells were spun on glass slides, fixed with 4% PFA (Sigma-Aldrich, 20649296018) for 10 min at room temperature and washed three times for 5 min with washing buffer (Dako, Glostrup, Denmark, S3006). Afterwards, cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, T8787) in PBS for 10 min at room temperature (RT) and washed three times for 5 min with washing buffer. DAKO Protein Blocking Solution (Dako, X0909) was added and incubated for 1 h at RT. Cells were subjected to immunofluorescence staining with primary antibodies specific for PGRMC1 (Abcam ab48012), ERα (Abcam ab259427), PHB1 (Abcam ab75766) and PHB2 (Cell signaling 14085S) overnight at 4 °C. Afterwards, the slides were washed three times for 5 min with washing buffer and a respective fluorophore labeled secondary antibody (anti-goat: Invitrogen, A11055; anti-mouse: Invitrogen, 745480; anti-rabbit: Invitrogen, A31573) was added to the samples and incubated for 1 h at RT in the dark. The slides were washed three times for 5 min with washing buffer and incubated with DAPI (Thermo Fisher Scientific, 15733122) for 5 min at RT. Antibody incubation steps were performed in a humidified chamber. The slides were washed with distilled water, mounted with Fluorescent Mounting Medium (Dako, S3023) and dried overnight. The cells were examined by fluorescence microscopy using the Axioplan 2 Imaging fluorescence microscope.