Acyl-biotin exchange assay (ABE)

Palmitoylation levels were determined as previously described (Thomas et al., 2012). 8 hr after transfection HEK293T cells were lysed and sonicated in ABE lysis buffer (50 mM HEPES pH 7.0, 2% [w/v] SDS, 1 mM EDTA plus PIC) with 20 mM thiol-reactive methyl-methane thiosulfonate (MMTS, Thermo Fisher Scientific) to block free cysteine residues. Transduced hippocampal neurons were lysed and sonicated in the same lysis buffer on DIV16 and lysates passed through a QIAshredder homogenization column (#79654, Qiagen, Hilden, Germany). HEK293T cell and hippocampal neuron lysates were then incubated at 50°C for 20 min after which protein was precipitated and excess MMTS removed by acetone precipitation. Protein pellets were then dissolved in 4% SDS buffer (4% [w/v] SDS, 50 mM Tris pH 7.5, 5 mM EDTA plus PIC) and the resultant solution wassplit in two and incubated for one hour with either 0.7 M hydroxylamine pH 7.4 (+NH₂OH, HAM, Thermo Fisher Scientific), to cleave thioester bonds and remove palmitate groups, or with 50 mM Tris pH 7.4 (buffer control, -NH₂OH). Both buffers contained 1 mM biotin-HPDP (Soltec Ventures, Beverly, MA) to biotinylate newly revealed cysteines. Protein was then acetone precipitated again to remove excess hydroxylamine and biotin-HPDP and pellets were re-dissolved in lysis buffer plus PIC without MMTS and then diluted 1:20 in dilution buffer (50 mM HEPES, 1% [v/v] Triton X-100, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl plus protease inhibitors). Biotinylated proteins were then purified using high capacity neutravidin-conjugated beads (Thermo Fisher Scientific) via incubation for three hours at 4°C after which beads were washed extensively in dilution buffer plus 0.5M NaCl. Purified protein was then eluted by HPDP reduction with 1% (v/v) β-mercaptoethanol (Millipore-Sigma) in elution buffer (0.2% [w/v] SDS and 0.25M NaCl in dilution buffer) for 10 min at 37°C. Eluted protein was then denatured in SB and subjected to SDS-PAGE followed by western blot as described below.