Relative activities of cytosolic (top) and membrane fractions (bottom) of HEK293 cells transfected with eYFP-PLCβ1wt, eYFP-PLCβ1 S887A, eYFP-PLCβ1 S887D or free eYFP. Membrane fractions were prepared as described in the methods. Enzyme concentrations were normalized by eYFP fluorescence and 60 μL of solution was incubated at 30°C with 2mM small unilamellar vesicles consisting of POPC:POPS:POPE:PIP2 31:32:32:5 doped with 3H-PIP2 at 60 μM free Ca2+, The amount of soluble 3H-Ins(1,4,5)P3 product was then measured after 3 minutes, where n=6-9 and S.D.is shown.