Fig. 1

Analysis of the interaction of TF and MAGI1-3 by proximity ligation assay and the influence of PAR2 activation. MDA-MB-231 cells (10³) were seeded out into 35 mm-glass based μ-dishes and adapted to serum-free medium for 1 h prior to activation. The cells were then incubated with PAR2-AP (20 μM) for up to 30 min and then fixed with 4% (v/v) paraformaldehyde for 15 min. The cells were washed three times with PBS and permeabilised with Triton X-100 0.1% (v/v) in PBS, for 5 min. All samples were blocked with Duolink blocking buffer for 1 h and incubated overnight with combinations of antibodies as follows, at 4 °C. The proximity between TF and MAGI1-3 were examined using a mouse anti-TF antibody HTF1 (5 μg/ml) together with a rabbit anti-MAGI1 antibody (H-70; 2 μg/ml), a rabbit anti-MAGI2 antibody (2 μg/ml) or a rabbit anti-MAGI3 antibody (2 μg/ml). The antibodies were diluted in the provided antibody diluent and blocked with the provided blocking buffer. The cells were washed three times with PBS and PLA performed according to the manufacturer’s instructions. The cells were labelled with DAPI (2 μg/ml) and Phalloidin-FITC (2 µg/ml). Images were acquired using a Zeiss Axio Vert.A1 inverted fluorescence microscope with a × 40 magnification. (The micrographs are representative of 5 fields of view from 9 experiments, RED = PLA incidences; GREEN = Phalloidin; BLUE = DAPI). B The interactions of TF with MAGI1-3 in non-activated and at 20 min post-activation was analysed. C The interaction of TF and MAGI1 at intervals up to 40 min was analysed by PLA