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. 2020 Apr 22;10(4):647. doi: 10.3390/biom10040647

Figure 3.

Figure 3

The cPLA2α C2 domain oligomerizes on lipid vesicles. (A) schematic of the crosslinking assay displaying the possible experimental outcomes. First, C2 domain with label is highlighted in the left panel of A, followed by the C2 domain binding to vesicles (POPC in blue), which would have captured oligomers in the presence of crosslinker (black line). Alternatively, at less protein density on the membrane surface, as in the presence of 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] nickel salt (DGS-NTA) (right panel of A), C2 oligomers are proposed to be in lower quantity. (B) 35 μL of 1.5 mM 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) large unilamellar vesicles (LUVs) were incubated with 8 μg of cPLA2α-C2 in 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4), 160 mM KCl and 50 μM CaCl2 for 30 min. The protein was crosslinked using 0.5 mM ethylene glycol bis(succinimidylsuccinate) (EGS) for 5 min, then the reaction was quenched with 150 mM Tris (pH, 8.0) for 5 min. The samples were run on 12% SDS-PAGE, stained with Coomassie blue, destained for 120 min, and analyzed with ImageJ. (C) Quantification of crosslinking experiments was completed in triplicate and normalized by dividing the >14-mer band density to the monomer band density as determined with ImageJ. (D) POPC or 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] nickel salt (DGS-NTA-Ni) lipids were incubated with buffer either containing 50 μM CaCl2 or 100 μM ethylene glycol-bis(β-aminoethyl)-N,N,N’,N’-tetraacetic acid (EGTA) to chelate calcium. Each mixture was resolved using SDS-PAGE, stained with Coomassie blue and destained. (E) A representative quantification of D, which was performed independently in duplicate. Data was normalized as stated in C. (F) A549 cells were transfected at 70–90% confluency with either mEGFP-cPLA2α-E116K or mEGFP-cPLA2α-D43N and imaged 24 h post transfection. Cells were treated with 10 μM A23187 for 30 min and representative images are shown.