FIGURE 6.

PI(4,5)P₂ binding of full-length DGKη and its PH-deleted mutant. A, 3xFLAG-tagged full-length DGKη1 (splice variant 1) and DGKη1-ΔPH (amino acids 164–1164) were expressed in COS-7 cells. The Western blotting analysis was performed using anti-FLAG antibody. B, equimolar amounts (100 pmol) of PI, PI(3)P, PI(4)P, PI(5)P, PC, PI(3,4)P₂, 18:0/20:4-PI(4,5)P₂, PI(3,4,5)P₃, PA, and PS were spotted onto nitrocellulose membranes as indicated. The membranes were incubated with COS-7 cell lysates containing 3xFLAG-tagged full-length DGKη or its PH deletion mutant. C, the blots were scanned and quantified using ImageJ software. The PI(4,5)P₂ binding level of full-length DGKη1 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 full-length DGKη was determined using Student's t test (**, p < 0.01; ***, p < 0.005). Error bars represent S.E.