Fig. 2

Analysis of the interaction of TF and MAGI1-3 and the influence of PAR2 activation. A MDA-MB-231 cells (2 × 10⁵) were adapted to serum-free medium for 1 h prior to activation. The cells were then incubated with PAR2-AP (20 μM) for up to 20 min and TF was immunoprecipitated from cell lysates with the anti-TF (HTF-1; 4 µg) antibody using protein A-magnetic beads. The samples were washed five times with PBST (1 ml) and denatured in SDS-PAGE loading buffer and examined for MAGI1 and TF by western blot using an anti-MAGI1 (H-70) and a rabbit anti-TF antibody (FL-295). B The ratio of the MAGI1 band densities were normalised against those of TF in the same co-immunoprecipitated samples. C In addition, MAGI1 was immunoprecipitated from cell lysates with an anti-MAGI1 (H-70; 4 µg) antibody using protein A-magnetic beads. The samples were washed five times with PBST (1 ml) and denatured in SDS-PAGE loading buffer and examined for TF and MAGI1 by western blot using an anti-TF antibody (HTF-1) and a mouse anti-MAGI1 antibody (SS-5). D The ratios of the TF band densities were normalised against those of MAGI1 in the same co-immunoprecipitated samples