Fig. 4

Examination of influence of TF phosphorylation on the binding of MAGI1 and 3. Biotinylated peptides, corresponding to the last 18 amino acids of the cytoplasmic domain of TF were synthesised in non-phosphorylated, single-phosphorylated and double-phosphorylated forms and used as bait. An additional scrambled peptide (biotin-SWGNVSKLSAPRQGVNKE) was also included alongside. The peptides (5 µM final concentration) in PBS, were bound into a NeutrAvidin-coated 96-well plate and then blocked. Cell lysates from resting and PAR2-activated MDA-MB-231 cells (from 2 × 10⁵ cells) were incubated in the plates for 1 h at room temperature. The wells were then washed four times and probed with (A) rabbit anti-MAGI1 (H-70) or (B) mouse anti-MAGI3 (46) antibodies diluted 1:200 (v/v) in PBST. The samples were detected using goat anti-rabbit and goat anti-mouse alkaline phosphatase-conjugated antibodies diluted 1:200 (v/v) and the colour developed with TMB One Solution (100 µl). Once the colour was developed the reactions were stopped and absorptions recorded. (n = 3; * = p < 0.05 vs. the respective samples without cell lysate). C The interaction of MAGI1 with the non-phosphorylated TF peptide was examined by pull-down over a period of 30 min following activation of PAR2, as described above