Fig. 6. CrPRF has a low affinity for PtdIns(4,5)P2. (A) Microfiltration of profilin-PtdIns(4,5)P2 complexes shows that little CrPRF bound to lipid micelles. The indicated concentrations of PtdIns(4,5)P2 in micelles were incubated with 2.5 µM profilin and spun through a 30,000 molecular weight cut-off filter. The flow through was analysed by SDS-PAGE. The 14-kDa region of gels from a representative experiment are shown for CrPRF, ZmPRO5 and HPRO1 (top). The intensity of each Coomassie-stained band was determined with a densitometer and normalized against the intensity of the profilin band found in the flow through in the absence of PtdIns(4,5)P2. Bars (bottom) represent the percentage of CrPRF (black), ZmPRO5 (grey) or HPRO1 (white) present in the flow through from two independent experiments. (B) The hydrolysis of PtdIns(4,5)P2 by phospholipase C (PIC) was measured in the absence or presence of 5 µM profilin. Each bar represents the average (± standard deviation) of at least four independent determinations. PIC activity in the absence of profilin (ø profilin) was set to 100%.
