Fig. 6. SigD depletes plasma membrane PtdIns(4,5)P₂ and inhibits mast cell degranulation. (A) RPMCs were permeabilised with SL-O in the presence of 100 µM MgATP and 300 µM Ca:EGTA at pCa 8 in the absence (Ctrl) or presence of 100 µg/ml of SigD wild-type or the inactive SigD^C462S mutant. After 10 minutes at 30°C, cells were fixed and stained with 2C11 and Alexa Fluor 488-phalloidin. Top panels show phalloidin staining, bottom panels show the fluorescence intensity profile for 2C11. Bar, 10 µm. (B) Mast cells in suspension were treated as in A. After 10 minutes at 30°C, cells were kept inactive by addition of 3 mM Ca:EGTA at pCa 7, or stimulated with 3 mM Ca:EGTA at pCa 5 and 100 µM GTPS (SigD^C462S) as indicated. After a further 10 minutes, cells were quenched with ice-cold 5 mM EGTA, centrifuged and supernatants assayed for ß-hexosaminidase (ß-hex) activity.