Figure 4

DdRACK1 binds to phosphoinositides. (A) PIP-Strip-membranes were incubated for 1 h at room temperature with 1 μg/ml GST (control), GST-DdRACK1 and GST-DdRACK1mut, respectively. Binding to PIPs on membranes was detected by incubation with polyclonal anti-GST antibodies. (B) Binding of GST-DdRACK1, GST-DdRACK1mut and GST (control) to PIPs in a liposome binding assay. 5–10 μg of GST and the GST-fusion proteins were incubated with liposomes containing 10% (wt/wt) of the indicated PIPs. Liposomes were collected by centrifugation, and bound proteins resolved by SDS-PAGE and detected by Coomassie Blue staining (S = supernatant; P = pellet). (C) Quantification of bound GST-DdARCK1 and GST-DdRACK1mut in pellet samples from (B). Protein bands were quantified with ImageJ software.