Ab50391

Following transfection, MDA-MB-231 cells cultured on glass coverslips with different cells surfaces were washed with PBS supplemented with Ca2+ and Mg2+, then fixed for 20 min with 4% PFA (diluted in PBS) at room temperature. The cells were subsequently washed 3 times with PBS (10 min each), followed by a 10 min permeabilization with 0.5% Triton-X 100 (diluted in PBS). After 30 min of blocking-10% goat serum (diluted in 0.5% Triton-X 100), the cells were incubated for 1 h with either an antizyxin antibody (Abcam; ab50391) (1:100) or antipaxillin antibody (Abcam; ab23510) (1:100), both diluted in 2% goat serum. Excess of primary antibodies was removed by 3 sequential washes with PBS (10 min each); thereafter, the cells were incubated for 1 h with an Alexa Fluor 488 conjugated to a rabbit anti-IgG antibody (Cell Signalling; 4412; 1:100). After the removal of unbound secondary antibodies with 3 sequential washes (PBS; 10 min each time), the nuclei were stained, and the cells were mounted onto glass slides with the Fluoroshield mounting medium containing DAPI. The cells were visualised by confocal microscopy (Nikon Eclipse Ti Laser-scanner). The DAPI was detected at 350 nm/470 nm (excitation/emission) while the Alexa Fluor 488 was detected at 488 nm/510 nm (excitation/emission).

Forskolin (F6886), epinephrine (E4642), phorbol-12-myristate-13-acetate (P1585), heparin (H3149), 4′, 6-diamidino-2 phenylindole dihydrochloride (DAPI, D8417), dimethylsulfoxide (D2650) and anti-flag M2 affinity gel (A2220) were from Sigma. Rabbit anti-VWF antibody (A0082) was from Dako. Rabbit polyclonal antibody to ERK2 (sc-292838) was from Santa Cruz Biotechnology. Rabbit and mouse monoclonal antibodies against zyxin (ab109316, ab50391), mouse monoclonal antibodies to vinculin (ab18058) and mouse monoclonal antibody to mouse α-actinin (ab18061) were from Abcam. Mouse monoclonal antibody to human P-selectin (Q01102) was from R&D Systems. Rabbit polyclonal antibody to phospho-zyxin Ser 142/143 (#4863) was from Cell Signaling Technology. Alexa Fluor 488-nm-conjugated phalloidin (A12379) and Alexa Fluor-conjugated goat anti-IgG antibodies (A11008, A27039, A21424, A21052) were from Invitrogen.