1 2 diacyl sn glycero 3 phospho l serine

Caffeic acid (3-(3,4-dihydroxyphenyl)-2-propenoic acid, CFAH₃), p-coumaric acid (4-hydroxycinnamic acid, p-CAH₂), Eu(III) chloride hexahydrate (EuCl₃·6H₂O), Dy(III) chloride hexahydrate (DyCl₃·6H₂O), and Gd(III) chloride hexahydrate (GdCl₃·6H₂O), DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), potassium persulfate (K₂S₂O₈), TPZT (2,4,6-tris(2-pyridyl)-s-triazine), iron(III) chloride hexahydrate (FeCl₃∙6H₂O), iron(II) sulfate (FeSO₄), potassium bromide (KBr), gentamycin and fluconazole were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Phospholipids, namely 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (16:0–18:1 PG, POPG, purity > 99%), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0–18:1 PE, POPE, purity > 99%), 1,2-diacyl-sn-glycero-3-phospho-L-serine (PS, purity > 97%), dipalmitoylphosphatidylcholine (16:0 PC, DPPC, purity > 99%), and E. coli polar lipid (EPL) extract, were also purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium hydroxide (NaOH) was purchased from POCH S.A. (Gliwice, Poland). Methanol was sourced from Merck (Darmstadt, Germany). Mueller-Hinton agar was supplied by Oxoid (Hampshire, UK). All chemicals had an analytical purity and were used without further purification.

Lipids from samples of DRM fractions were extracted as in the case of TLC. Then, lipids were spotted onto a Hybond-C membrane. The membrane was subsequently air-dried, blocked with 1% defatted milk in PBS for 1 h and overlaid with 2.5 μg/mL of recombinant toxin in blocking buffer overnight. After three washes with PBS supplemented with 0.1% Tween 20, bound toxin was detected using a polyclonal specific IgG raised in rabbit [17 (link)] and subsequent chemiluminescence, as in the case of western blots. To determine the presence of sulfatide in DRM gradients, a monoclonal antibody against sulfatide was used [51 (link)]. In some analyses, commercial membranes with spotted lipids were also used (Membrane lipid strips and PIP strips from Echelon Biosciences, USA) or, alternatively, commercial lipids were spotted onto Hybond C membranes. These lipids were: 1,2-Diacyl-sn-glycero-3-phospho-L-serine, sulfatide and a phosphoinositide mix (PIs) from bovine brain (all from Sigma). When indicated, signal intensity was determined using the GeneTools software (Syngene; Cambridge, UK).

Soybean-phosphatidylcholine (PC, 95%) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Tributyrin (TC4, 99%), Tricaprylin (TC8, 99%), Triolein (TC18, 99%), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, ≥97%), l-α-phosphatidylinositol (PI, ≥99%), 1,2-diacyl-sn-glycero-3-phospho-l-serine (PS, ≥97%), l-α-phosphatidylethanolamine (PE, ≥99%), cardiolipin (CL, ≥98%), 3-sn-Phosphatidic acid sodium salt (PA, ≥98%), Sphingomyelin (SM, ≥98%), 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (PG, ≥98%), 1,2-distearoyimonoglactosylglyceride (MGDG, ≥98%) were all from Sigma-Aldrich Corporation (St. Louis, MO, USA). Olive oil and sucrose esters were purchased from Aladdin Reagent Co. (Shanghai, China). Six pure chiral pseudoglycerides (didecanoyl-deoxyamino-O-methyl glycerol, DDGs) used in the present studies were synthesized according to the method reported before [23 (link)].