NEC Sensitivity to Membrane Curvature

NEC sensitivity to membrane curvature was tested using coflotation as described previously (120 (link)). Briefly, 1.5 μg NEC was incubated with or without large unilamellar vesicles (LUVs) (POPC, POPS, and POPA mixed in a 3:1:1 molar ratio as previously described [21 (link)]) at room temperature for 20 min in 50 ml phosphate-buffered saline (PBS). KCl was added to 200 mM concentration to reduce nonspecific protein-membrane interactions, and samples were incubated for 15 min at room temperature. OptiPrep (Sigma) was added to a final concentration of 30% in a 500-ml volume. Samples were placed at the bottom of a 5-ml centrifugation tube (Beckmann) and overlaid with 4 ml 15% OptiPrep and 500 ml 3% OptiPrep in PBS. The samples were next centrifuged in a Beckman SW-55 Ti rotor at 246,000 × g for 3 h at 4°C, and 1-ml fractions were collected beginning at the top. Protein was precipitated with 20% trichloroacetic acid for 30 min on ice. Sample was washed with 750 μl cold acetone and then spun in a tabletop centrifuge for 10 min at 14,000 rpm. This was repeated for a total of 3 washes. Samples were then analyzed by Western blotting for UL31 as previously described (21 (link)). The standard error of the mean is reported from at least two individual experiments. Data were plotted using GraphPad Prism 9.0.