Figure 6
InsP 3 and PtdIns(4,5)P 2 binding of recombinant PLCδ1PH-GFP and GFP-Tubby-domains. Panel A: InsP3 binding assays were performed at room temperature using [3H]-Ins(1,4,5)P3 in the presence or absence of the indicated concentrations of unlabeled InsP3 as detailed under "Experimental Procedures". Means ± range of 2 experiments performed in duplicate are shown. Panel B shows the binding of recombinant proteins to lipid vesicles containing phosphatidyletanolamine in the absence or presence of PtdIns(4,5)P2 and InsP3. Vesicles were pelleted with ultracentrifugation and ''S'' and ''P'' represent the soluble (unbound) and pellet-associated (bound) GFP fusion protein, respectively, analyzed by a PhosphorImager after SDS-PAGE. The result of one of two experiments with identical results is shown. Note the negligible InsP3 yet very good PtdIns(4,5)P2 binding of the Tubby domain. The latter shows only a minor sensitivity to the presence of InsP3 in contrast to PLCδ1PH-GFP.