JCS02912 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Mast cells contain a nuclear detergent-resistant pool of PtdIns(4,5)P₂. Mast cells were fixed with 4% paraformaldehyde at 25C either at rest or after stimulation with 48/80 for the indicated times. Subsequently, cellular membranes were extracted with 0.2% Triton X-100. (A) Top panels show cells stained with Draq5 (blue), 2C11 (red) and Alexa Fluor 488-phalloidin (green); centre panels show an enlarged view of the nuclei indicated with an asterisk. Bottom panels show 2C11 fluorescence intensity profiles for the nuclei from centre panels. Bar, 10 mm. (B) Mean 2C11 fluorescence for nuclei (n>100) plotted against duration of stimulation with 48/80, and are s.e.m.

• Supplemental Figure 2 -

  Fig. S2. Depletion of plasma membrane PtdIns(4,5)P₂ from cells labelled for 19 hours in vitro with inositol as described in Fig. 2D. (A) Cells were fixed with ice-cold 3% glutaraldehyde and stained either at rest, or after stimulation for the indicated times with compound 48/80. Upper panels show merged images of 2C11 (red) and Alexa Fluor 647-concanavalin A (blue); lower panels show fluorescence intensity profiles for 2C11. Bar, 10 mm. Ctrl indicates that 2C11 has been omitted during staining. (B) 2C11 fluorescence was quantified as described in Fig. 2B-D; the distribution of fluorescence at the indicated time points is shown as in Fig. 2C.

• Supplemental Figure 3 -

  Fig. S3. Concentration-dependent inhibition of mast cell exocytosis by PLC inhibitors. RPMCs in suspension were permeabilised in the presence of 100 mM MgATP and 3 mM Ca:EGTA at pCa 7 (n=1), or 100 mM MgATP, 100 mM GTPgS and 3 mM Ca:EGTA at pCa 5 (n=3) in the presence of the indicated concentrations of U73-122 or U73-343 (A) and Et-18-OMe or EtOH (B) at 30C. After 10 minutes, secretion was quenched and released b-hex activity assayed as described in Fig. 6B.

• Supplemental Figure 4 -

  Fig. S4. PtdIns-PLC does not affect PtdIns(4,5)P₂ levels in permeabilised RPMCs. Adherent mast cells were permeabilised for 10 minutes in the presence of 100 mM MgATP and 300 mM Ca:EGTA at pCa 8, with (B) or without (A) 0.3 U/ml PtdIns-PLC. Cells were then fixed and stained with 2C11. Fluorescence intensity profiles are shown.