N octylglucoside

This assay is based on previously described three-step chemical method where free unmodified cysteine thiols are blocked with NEM (N-ethyl maleimide) followed by palmitoylation thioesters cleavage by hydroxylamine (NH₂OH); finally, loading thiol-specific biotinylating reagent (HPDP-biotin in our experiments) to the newly exposed cysteinyl thiols. Biotinylated proteins are then affinity purified with streptavidin–agarose beads and probed for the protein of interest (Bhattacharyya et al., 2013 (link)). The assay was performed on total cell lysates extracted from differentiated FAD NPCs. In some cases, cells were treated with sigma1 receptor (S1R) agonist PRE-084 or antagonist NE-100 before ABE assay. ABE assays were also performed on cells undergoing cycloheximide (CHX) chase for 0–18h. For lipid raft, MAMs and non-raft palmitoylation studies, protein extracts from lipid rafts, MAMs and non-rafts were subjected to ABE assay according to a method reported previously (Bhattacharyya et al., 2013 (link)). Briefly, lipid raft and MAM fractions were extracted with 60 mM n-Octylglucoside (Sigma), and non-raft fractions were extracted with 1% Triton X-100. The protein extracts were precipitated using chloroform/methanol before ABE assay.

To prepare the liposome, the detergent remove method was used. Cholesterol (Sigma Aldrich, USA), L-alpha phosphatidylcholine (PC) (Sigma Aldrich, USA), biotinyl phosphoethanolamine (biotin-PE) (Avanti Polar Lipids, USA), and Ni-NTA-DGS (Avanti Polar Lipids, USA) were first dissolved in 60 ˚C N-Octylglucoside (Sigma Aldrich, USA) solution to make lipid stock solutions. The liposome for the experiment was generated by mixing the lipids with a ratio of PC: Cholesterol: Ni-NTA-DGS: biotin-PE = 16:2:2:1, to achieve a total lipid concentration of 800 mM. After mixing, 540 ul Tris-buffered saline (0.01 M, pH 7.4, 150 mM NaCl) was added to the mix and followed by eliminating N-Octylglucoside with the Pierson detergent removal column (Thermo Fisher, USA). Then, the detergent-free lipid mix was vortex and extruded through 0.2 μm syringe filter at 60 ˚C for 15 times to construct the liposome with unified size.
To functionalize the liposomes, the His-tagged human recombinant E-selectin (10335-H03H, Sino Biological, USA) was used. 45 μl of 0.58 μM E-selectin solution was added to the liposome solution. To collect the E-selectin conjugated liposome, the solution was centrifuged at 100,000xg for 3 hours at 4 °C. After the centrifuge, the supernatant was removed, and the pellet was resuspended in 100 μl Tris-buffered saline. The liposome samples were stored at 4 °C and used within 24 hours.