, Hill equation used for cooperative ligand-receptor binding. Size Exclusion Chromatography-Multi-Angle Light Scattering

, Purified M13-cpFAST2-CaM (3.5 mg at 113 µM) was injected in a Superdex 200 Increase (GE Lifesciences) previously equilibrated in 10 mM HEPES, 50 mM NaCl, 2 mM CaCl2, pH 7.5 connected to a TREOS static light detector and to a t-Rex refractometer (both purchased from Wyatt Technologies). Molecular mass was determined by the Astra software (Wyatt)

, at 37 °C in a 5% CO2 atmosphere. For imaging, cells were seeded in ?Dish IBIDI (Biovalley) coated with poly-L-lysine. Cells were transiently transfected using Genejuice (Merck) according to the manufacturer's protocol for 24 hours prior to imaging. calcium imaging, cells were washed twice with Dulbecco's PhosphateBuffered Saline (DPBS) immediately prior to imaging. After rinsing, solution of fluorogen in HHBSS, Mammalian cell culture HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with phenol red, Glutamax I, and 10% (vol/vol) fetal calf serum (FCS)

, Approximately 2 min after the start of the experiment, histamine (50 ?M final concentration) in HHBSS containing the appropriate amount of fluorogen to maintain it constant was added to allow histamine-induced oscillations to subside

, Image J using a line ROI with a width of 15 pixels. Presented images are a maximum projection of 10 raw images while the movies were generated as a walking average of 5 frames

M. Plamont, E. Billon-denis, S. Maurin, C. Gauron, F. M. Pimenta et al., Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo, Proc Natl Acad Sci, vol.113, pp.497-502, 2016.
URL : https://hal.archives-ouvertes.fr/hal-01259909

C. Li, M. Plamont, H. L. Sladitschek, V. Rodrigues, I. Aujard et al., Dynamic multicolor protein labeling in living cells, Chem Sci, vol.8, pp.5598-5605, 2017.

R. Tsien, P. , and T. , Measurement of cytosolic free Ca 2+ with quin2, Methods in Enzymology, pp.230-262, 1989.