FIGURE 4.

Lipid binding activities of DsRed monomer-tagged DGKη-PH and PLCδ1-PH expressed in COS-7 cells. A, expression of DsRed monomer alone, DsRed monomer-DGKη-PH, and DsRed monomer-PLCδ1-PH was confirmed by Western blotting. B, equimolar amounts (100 pmol) of PI, PI(3)P, PI(4)P, PI(5)P, PC, PI(3,4)P₂, PI(4,5)P₂, and PI(3,4,5)P₃ were spotted onto nitrocellulose membranes as indicated. The membranes were incubated with DsRed monomer-tagged DGKη-PH and PLCδ1-PH. Lipid-bound DsRed monomer-tagged proteins were detected by immunostaining with anti-RFP antibody, which cross-reacts with DsRed monomer. C, the blots were scanned and quantified using ImageJ software. The PI(4,5)P₂ binding level of DGKη-PH was set to 100%. The data are shown as the means ± S.D. of three independent experiments. Statistical significance compared with the PI(4,5)P₂ binding activity of DGKη-PH was determined using Student's t test (***, p < 0.005). Error bars represent S.E.