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Since isolation of pollen tube DIMs was carried out from the microsomal fraction, the presence of mitochondria and plastid proteins could otherwise depend on the presence of mitochondrial/plastid DIMs. To answer this question western blot analysis was performed on P2 and DIMs, using antibodies against the mitochondria and plastid markers cytochrome-c oxidase subunit II (COXII) and glutamine oxoglutarate aminotransferase (GOGAT), respectively. The anti COXII antibody recognised a band with a molecular mass of about 30?kDa both in P2 and DIMs (Fig.?6Aa), suggesting that mitochondria were present in the microsomal [https://en.wikipedia.org/wiki/Sitaxentan Sitaxentan] fraction and that mitochondrial DIMs were present in our preparation. On the contrary, while plastids appeared to be present in P2, no reaction [http://www.selleckchem.com/products/LBH-589.html www.selleckchem.com/products/LBH-589.html] was detected in DIMs (Fig.?6Aa), supporting the idea that plastid membranes do not contaminate DIMs and that the presence of plastid proteins could be due to spurious contamination by proteins that are solubilised during the DIM isolation procedure. Fig. 6. DIM characterisation. Among mitochondrial proteins enhanced in DIMs, MALDI-TOF MS and LC-MS/MS analysis identified at least four mitochondrial VDACs/porins, that were also found in DIMs isolated from Medicago truncatula PM (Lefebvre et al., 2007). To verify the localisation of VDACs in pollen tubes we performed immunolocalisation experiments using a polyclonal antibody against VDACs. The specificity of the antibody was previously tested on the pollen tube crude extract (Fig.?6Ab). According to MALDI-TOF MS and LC-MS/MS analysis, the anti-VDAC antibody recognised bands between 40?kDa and 29?kDa. Immunogold-labelling experiments revealed that VDACs localised on the outer mitochondrial membrane (Fig.?6Ba,b, arrows) and decorated cytoplasmic vesicles (Fig.?6Ba, arrowheads). Control experiments [http://www.selleckchem.com/products/Paclitaxel(Taxol).html Paclitaxel concentration] in which the primary antibody was omitted showed a few, occasional gold particles dispersed in the cytoplasm (supplementary material Fig. S5b), whereas cytoplasmic organelles did not stain (supplementary material Fig. S5a�Cc). ��-cyclodextrin dramatically inhibited the pollen tube growth and affected both DIMs and pollen tube morphology To validate our data showing lipid microdomains in Angiosperm pollen tubes, we investigated the effect of different concentrations of ��-cyclodextrin (BCD) on DIMs and on pollen tube morphology. The BCD is known to extract sterols from membranes and to destroy lipid rafts in different cell types (Ohtani et al., 1989; Ilangumaran and Hoessli, 1998; Roche et al., 2008). In order to observe the effects of sterol deprivation on isolated DIMs and on pollen tube ultrastructure, tobacco pollen tubes were incubated with 8?mM or 16?mM BCD. Germination assays were performed to identify the best BCD concentration and incubation time. We found that incubation with both 8?mM and 16?mM BCD for 2?h and 30?min, dramatically inhibited pollen tube elongation (supplementary material Fig. S6a�Cd; P