Figure 2

The PH domain binds to phosphoinositides. ( A ) PIP-Strip-membranes were incubated over night with GST-PH domain (1 μg/ml) and with GST (1 μg/ml) for control. Binding was detected by incubation with polyclonal GST-antibodies. ( B ) Binding of Sec7-PH domain (residues 454-577) to PIs in a liposome binding assay. Upper panel, 5 μg of the GST-PH fusion protein were incubated with liposomes containing 10% (wt/wt) of the indicated PIs. Liposomes were collected by centrifugation, and bound proteins resolved by SDS-PAGE and detected by Coomassie Blue staining (S = supernatant; P = pellet). Lower panel, for quantification, the bands were scanned and the ratio of the pellet vs. supernatant intensities of each PI fraction after background subtraction calculated. Error bars, SD of liposome binding from two independent experiments. ( C ) Binding of the Sec7 PH domain to PI(3)P, PI(4)P, PI(5)P and PI(3,5)P was negligible. ( D ) Liposome binding assay using GST for control.