Biotin peg4 alkyne

Streptavidin (bs-0437P, Bioss), Streptavidin-AF647 (bs-0437P-AF647, Bioss), SA-HRP (B110053-0100, Diamond), Biotin-XX Tyramide (A8012-10, APExBIO), a BCA Protein Assay Kit (PT0001, Leagene), ProLong Gold Antifade Mountant with DAPI (P36941, Invitrogen), EdU (ST067, Beyotime), afatinib (BD210970, Bide Pharmatech), Biotin-PEG₄-alkyne (#764213, Sigma), and BTTAA (BDJ-4, Confluore) were used.

Visualization of the palmitoylome in growth cones or F11 cells using bioorthogonal labeling and click chemistry was adapted from protocols as detailed elsewhere (Gao and Hannoush, 2014 (link)). Briefly, embryonic DRG or F11 cells were grown on PDL/laminin1 coated plates overnight and then further cultivated in culture medium supplemented with 100 μM 15-azidopentadecanoic acid (palmitic acid azide, Invitrogen, #C10265) or vehicle overnight. On the next day, cells were washed 3 times with pre-warmed PBS and then fixed 15 min at 37°C with pre-warmed 4% PFA in PBS. They were then washed 3 times 5 min in PBS at room temperature.
For click chemistry cells were permeabilized 4 min in PBS containing 0.1% Triton X-100 at room temperature and washed 6 times (5 min each) with PBS to remove the detergent. In each well, 100 μl of a solution containing 0.1 mM Biotin-PEG4-alkyne (Sigma, #764213), 1 mM TCEP [Tris(2-carboxyethyl)phosphine; Sigma, #C4706] and 1 mM CuSO₄ (Sigma, #C1297) in PBS were added and cells were incubated in the dark at room temperature for 1 h (see scheme of the click reaction in Supplementary Figure S5). Cells were then washed 6 times with PBS. Palmitoylated proteins were visualized by streptavidin-Cy5. The optimal concentration of palmitic acid azide was established in pre-experiments using F11 cells (see Supplementary Figures S5B₁–B₃).