Ez link hpdp biotin

The Acyl-Biotin Exchange was carried out as described in ref. 46 (link) with some modifications. Briefly, proteins from total cell homogenates were precipitated using chloroform:methanol (1:4 v/v) and treated overnight at 4 °C with 3 mM N-ethylmaleimide (NEM) (Sigma-Aldrich, St Louis, MO, USA) to block the free thiol groups. Free NEM was then removed by three sequential chloroform:methanol (1:4 v/v) precipitations, and samples were treated with 1 M hydroxylamine (Sigma-Aldrich, St Louis, MO, USA) and 1 mM EZ-Link HPDP-Biotin (Pierce Biotechnology, Waltham, MA, USA) for 1 h at room temperature to exchange thiol-bound fatty acids for biotin. In this step, omission of hydroxylamine served as a negative control. Samples were then incubated with streptavidin agarose beads (Pierce Biotechnology, Waltham, MA, USA) for 1 h at room temperature. All incubations were carried out in a buffer containing 50 mM Tris/HCl pH 7.0, 5 mM EDTA, 0.2% Triton X-100, 150 mM NaCl, 10 mM phenylmethanesulfonyl fluoride and PIC. Finally, all samples were centrifuged at 13,000 g for 1 min, unbound proteins were removed and the palmitoylated ones were eluted from the beads with 75 mM Dithiothreitol (DTT) for 10 min at 90 °C and processed for Western blotting. A commercial human Neu3 (Origene, Rockville, MD, USA) purified from HEK-293 cells was also subjected to ABE analysis.

Cells plated in 10-cm dishes were transfected with 5 μg of expression plasmids and then incubated for 24 h in complete medium containing 10 μM AACOCF3 and either 1 μM palmostatin B or DMSO vehicle (0.1%). Cells were lysed in 1 mL of lysis buffer (50 mM Tris–HCl, pH 7.2, 1% Triton X-100, 150 mM NaCl, 1% sodium deoxycholate, 1 mM EDTA, 0.1% SDS, and protease and phosphatase inhibitors containing 10 mM N-ethylmaleimide) (Sigma). Acyl-biotin exchange assays were performed as described previously with minor modifications [36 (link)]. Briefly, 5 μg of protein were precipitated using chloroform-methanol and treated exhaustively with N-ethylmaleimide to block free thiol groups, which was removed by sequential chloroform-methanol precipitation. The samples were then treated with hydroxylamine (Sigma) and EZ-Link® HPDP-Biotin (Pierce Biotechnology, Rockford, IL, USA) to exchange thiol-bound fatty acids for biotin. Equal amounts of solubilized protein were incubated with 15 μL of streptavidin agarose resin (Pierce Biotechnology) at 25°C for 90 min. Immunoprecipitated complexes were analyzed by Western blotting using an anti-RAS antibody after washing three times with washing buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.2% Triton X-100, and 0.1% SDS).

Poly-adenylated mRNA was purified using the Ambion Dynabeads mRNA Purification Kit (Ambion – 61006) according to the manufacturer’s protocol. Purified mRNA was fragmented for 4 min at 94 °C using the NEBNext Magnesium RNA Fragmentation Module (NEB – E6150S) to approximately 200–500 bases, recovered by ethanol precipitation, and dissolved in 50 μl RNase-free water. TU-tagged RNA was biotinylated using EZ-Link HPDP-Biotin (Thermo Scientific – 21341) by the addition of 25 μl 4x TE and 25 μl 1 mg/ml EZ-Link (dissolved in DMF) to the 50 μl RNA. Following a 3 h incubation at room temperature in the dark, excess biotin was removed by a chloroform/isoamyl alcohol (24:1) extraction as described [35 (link)]. The RNA was recovered by ethanol precipitation, at which point 80 ng of RNA was set aside as the “input” RNA sample.