Fig. 7

Outcome of expression of the MAGI1 N-terminal peptides on cell proliferation signalling. MDA-MB-231 cells (10⁵) were transfected to express the N-terminal of MAGI1 with and without the PDZ1 domain. Selected sets of cells were also pre-incubated with SAM11 antibody (20 µg/ml) as shown in the figures. The cells were lysed and proteins separated by denaturing 12% (w/v) polyacrylamide electrophoresis, transferred onto nitrocellulose membranes and blocked with TBST. A The membranes were probed with a goat anti-human Akt1/2 (N-19) polyclonal antibody and a rabbit polyclonal anti-human Akt1 (phospho-S473). The membranes were then washed and probed with a donkey anti-goat, or goat anti-rabbit alkaline phosphatase-conjugated antibody, diluted 1:4000 (v/v). Bands were visualised using the Western Blue stabilised alkaline phosphatase-substrate, recorded using ImageJ program and (B) the ratios calculated. C Separate sets of the western blot membrane were probed using an anti-phosphoT202/185-phosphoY204/187-ERK1/2 antibody or alternatively, total ERK1/2 was detected using an anti-ERK1/2 antibody diluted 1:3000 (v/v) in TBST. The membranes were also probed using a rabbit anti-GAPDH polyclonal antibody (V-18) diluted 1:5000 (v/v) in TBST. The membranes were then incubated with a goat anti-rabbit alkaline or a donkey anti-goat phosphatase-conjugated antibody diluted 1:5000 (v/v) in TBST and visualised as above, recorded using ImageJ program and (D) the ratios calculated. MDA-MB-231 cells (10⁵) were transfected to express the N-terminal of MAGI1 with and without the PDZ1 domain. Sets of cells were pre-incubated with a monoclonal antibody (SAM11; 20 µg/ml) to block PAR2 activation, or a mouse monoclonal antibody capable of inhibiting the protease activity of TF-fVIIa complex (HTF1; 20 µg/ml). E Total RNA was isolated from one set of the cells, and samples (100 ng) were amplified using the primers 5’- CCG TCC ATG CGG AAG ATC -3’ (forward) and 5’- ATG GCC AGC GGG AAG AC -3’ (reverse). The reaction was carried out at an annealing temperature of 60 °C using the GoTaq® 1-Step RT-qPCR for 40 cycles. Following amplification, the relative amounts of target mRNA were determined using the 2^−ΔΔCT method. F Cell numbers were determined in the second sets of cells using the crystal violet method