Figure 4
Identification of lipid rafts in S. mansoni.
(a) In situ detection of lipid rafts in control (-EGF) 24 h somules or 24 h somules/adult male worms treated with EGF (15 ng/ml; +EGF) for 5 min, stained live with the AlexaFluor 594 lipid raft labelling kit and imaged by confocal microscopy. (b) Identification of flotillin, Gq and Ras in adult worms (1 pair) and 24 h somules (~1000) by western blotting (with rat brain positive control) and in situ localization within somules by confocal microscopy using anti-flotillin, anti-Gq, and anti-Ras antibodies. The inset panels display localization of these proteins at the tegument region. (c) Detailed in situ analysis of PKC and ERK activation in 24 h somules exposed to EGF (15 ng/ml) using anti-phospho PKC (Thr410) and anti-phospho p44/42MAPK (Thr202/Tyr204) (ERK1/2) antibodies. (d) Separation of triton-insoluble (detergent-resistant) membranes from triton-soluble membranes and cytosolic fraction and identification of flotillin, Ras and tubulin within each fraction by western blotting; each lane contains equal amounts of total protein (39 μg) derived from fractionation of adult worm extracts. Representative micrographs of somules are single z scans. Bar = 25 μm.
