ITC experiments were performed using an
ITC-200 (Microcal). ITC
titrations performed in 20 mM Tris pH 7.4, 150 mM NaCl, 0.5 mM TCEP
(tris-carboxyethyl-phosphine) and 3 mM MgCl
2 at 25°C. Data
were integrated and fitted to a single-site binding equation using Origin 7
software with
integrated ITC module (Microcal).
Lilja J., Zacharchenko T., Georgiadou M., Jacquemet G., De Franceschi N., Peuhu E., Hamidi H., Pouwels J., Martens V., Nia F.H., Beifuss M., Boeckers T., Kreienkamp H.J., Barsukov I.L, & Ivaska J. (2017). SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras. Nature cell biology, 19(4), 292-305.