Biotin azide

Click chemistry palmitoylation assay was conducted imitating the method described by Coleman DT, et al. [15 (link)]. In brief, after the metabolic labeling the palmitoylated proteins with palmitate orthologs alkyne-linked 17-Odya (Cayman Chemical, USA), the cells lysates were further processed through the click reaction between biotin-azide (Cayman Chemical, USA) and alkyne-linked 17-Odya, which make the palmitoylated proteins covalently cross-linked with biotin at the S-palmitoylation sites. The biotinylated protein were pulled down from the cells lysates using streptavidin-agarose mini-column (Sigma, USA), and then were subjected to western blotting using monoclonal antibody recognizing EYFP (Amyjet Scientific, China).

The labeling protocol was adapted from [25 ]. In brief, to shed TNF-R1 from the cell surface and to trigger transport from the PM cells were washed in PBS and incubated in the presence of 150 μM histamine for 3 h in FCS free medium at cell culture conditions as adapted from Wang et al [26 (link)]. Histamine treated and untreated cells were then incubated for 16 h in the presence of 100 μM 17-ODYA (#90270, Cayman), followed by membrane sedimentation as described for acylRAC. The resulting pellet was resuspended in 150 μl 25 mM HEPES [pH 7.4], 25 mM NaCl, 0.5% Triton X-100, PIC. The click reaction was made up fresh with the final concentrations: 500 μM biotin-azide (#13040, Cayman)), 2 mM CuCO₄, 0.2 mM TBTA (#678937, Sigma Aldrich) and 4 mM ascorbic acid (fresh) in a total volume of 200 μl. After 2 h incubation at RT, proteins were acetone precipitated and then resuspended in 500 μl 50 mM TRIS-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 1% Triton X-100, 1 mM EDTA, 0.25% Na-deoxycholate. 20 μl Streptavidin-microbeads (#130–048-102, Miltenyi) were added and incubated overnight at 4 °C. After purification via μColums (Miltenyi) and elution using SDS-Sample buffer containing β-mercaptoethanol, 15 μl were used for SDS-PAGE/WB.