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. 2016 Feb 17;291(15):8150–8161. doi: 10.1074/jbc.M115.648717

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© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 11. — Lipid binding activities and subcellular localization of DsRed monomer-tagged DGKη-PH(K74A) and DGKη-PH(R85A) expressed in COS-7 cells. A, expression of DsRed monomer alone, DsRed monomer-DGKη-PH(K74A), and DsRed monomer-DGKη-PH(R85A) 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, and PI(4,5)P₂ were spotted onto nitrocellulose membranes as indicated. The membranes were incubated with DsRed monomer alone, DsRed monomer-DGKη-PH(K74A), and DsRed monomer-DGKη-PH(R85A). Lipid-bound DsRed monomer-tagged proteins were detected by immunostaining with anti-RFP antibody. Even after much longer exposure than that in Fig. 4, spots of PI(4,5)P₂ were not detected. DsRed monomer-DGKη-PH(K74A) (C) and DsRed monomer-DGKη-PH(R85A) (D) were co-expressed with AcGFP-PLCδ1-PH in COS-7 cells. After 48 h of transfection, the cells were incubated with 500 mm sorbitol in DMEM for 30 min. The cells were fixed with 3.7% formaldehyde and imaged by inverted confocal laser microscopy (FV1000-D). Representative data of three independent experiments are shown. Scale bars, 20 μm.