For PI-PLC treatment, lipids were dissolved in 0.1 ml of 50 mM Tris-Cl (pH 7.4) plus 0.16% (w/v) Triton X-100 and incubated for 1 h in the presence of 100 milliunits of Bacillus cereus PI-PLC (Sigma). All phospholipase reactions mixtures were partitioned between butanol and water,...
All other chemicals were purchased from Sigma (St Louis, MO). Lipid analysis Total lipids were extracted from the PG according to the method of Bligh and Dyer (13), and were separated by silca gel TLC. The plate was developed until 10 cm, in petroleum ether-diethyl ether-acetic acid (...
The cells were then permeabilized with 0.2% Triton X-100 (Sigma) in PBS for 15 min and blocked with 1% solution of bovine serum albumin BSA (Sigma) in PBS for 1 h. The plates were then incubated with the primary antibody followed by the secondary antibody for 1 h each with multiple ...