Fig. 5

Identification of the PDZ domain within MAGI1 responsible for binding TF. MDA-MB-231 cells (10³) were seeded out into 35 mm-glass based μ-dishes and separately transfected with constructs to express PDZ1-5 of from MAGI1. The cells permitted to express the proteins for 24 h and then fixed and washed. A The proximity of the expressed PDZ to TF was examined by PLA using a rabbit anti-HA (C2954) antibody and a mouse anti-TF (HTF1) antibody, respectively (RED = PLA incidences; GREEN = Phalloidin; BLUE = DAPI) and (B) quantified using the ImageJ program. C Cell surface TF-fVIIa activity was measured on the transfected cells expressing PDZ1, PDZ2 or the empty vector, using a modified thrombin-generation assay. An additional sample of cells were transfected to express PDZ1 but were pre-incubated with an inhibitory anti-TF antibody (HTF1; 20 µg/ml) before analysis. The cells (10⁵) were incubated for 20 min at 37 °C with a mixture of barium sulphate absorbable proteins (2 mg/ml) and 5 mM CaCl₂ in the reaction buffer (total volume 150 µl; Tris-buffered saline pH 7.4), containing 1% (w/v) bovine serum albumin (BSA). Aliquots of reaction (100 µl) were then transferred to 96-well plates containing 100 µl of thrombin substrate CS-01(38) (0.2 µM H–D-Phe-Pip-Arg-pNA) and incubated for a further 40 min at 37 °C. The reactions were stopped by the addition of 2% (v/v) acetic acid (50 µl) and the absorption of the sample measured at 410 nm on a plate reader