Fig. 1. PtdIns(4,5)P₂ is enriched at the plasma membrane of resting RPMCs. (A) The monoclonal anti-PtdIns(4,5)P₂ antibody 2C11 stains the plasma membrane. RPMCs were fixed and stained at 4°C with (a,b) 2C11 (red) and Draq5^TM (blue) or (c) CM-DiI as described in Materials and Methods; (a) shows an equatorial confocal section, (b,c) are projections of confocal sections taken at 1 µm intervals throughout the cell. (B) 2C11 binds PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃. 2C11 was pre-incubated with 1000-fold molar excess of the indicated inositol phosphate before staining as in (A); equatorial confocal sections are shown. (C) 2C11 specifically detects PtdIns(4,5)P₂ on the plasma membrane of RPMCs. Cells were preincubated with 50 µM of the indicated PH domain before staining (b,c), or with 1 mM neomycin during staining with 2C11 (a); equatorial confocal sections are shown. (D) PtdIns(4,5)P₂ colocalises with the cortical actin cytoskeleton. Cells were co-stained with 2C11 (red) and Alexa Fluor 488-phalloidin (green) as described in Materials and Methods. A projection of confocal sections taken at 1 µm intervals throughout the cell is shown. (A-C) Micrographs are at the same magnification, except (Ac), which is at the same magnification as (D). Bars, 10 µm.