Bovine intestinal alkaline phosphatase

In vitro enzyme assays were performed as previously described [63 (link), 64 (link)]. Briefly, a 400 μL reaction system containing 400 μM IPP and 200 μM FPP (100 mM HEPES, 5 mM MgCl₂, 10 mM KCl, pH 7.5) and 0.5 μmol/L of the purified recombinant fusion protein were incubated at 30 °C for 2 h. After inoculation, the solution was mixed with 200 μL of 0.2 mol/L Tris–HCl (pH 9.5) containing bovine intestinal alkaline phosphatase (20 mg/mL, more than 10 DEA units/mg, Sigma-Aldrich) and two units of shrimp alkaline phosphatase (1 unit/μL; TaKaRa). The reaction mixture was then incubated overnight at 30 °C to hydrolyse the diphosphate products into their corresponding alcohols. The mixture was extracted with hexane, and then three parallel samples of hydrolysate were concentrated to 100 µL in the experiment. Finally, the sample products were analysed using an LC–MS system equipped with an LTQ Orbitrap XL ETD analyser (Thermo Fisher Scientific, USA). The samples were separated using an Agilent 1200 Series HPLC system at a flow rate of 250 μL/min, and the mobile phase consisted of methanol (100%). The electrospray potential was 4.5 kV in positive electrospray ionisation (ESI) mode, and the source temperature was 275 °C. Three independent cultures were analysed for each set of experiments.

Immunoprecipitation of RIP1 was performed as described above. The immunoprecipitants were washed three times with a dephosphorylation buffer (DB; 100 mM NaCl, 50 mM Tri-HCl, 10 mM MgCl₂, 1 mM dithiothreitol, pH 7.9, and Sigma complete protease inhibitor set), then incubated in DB with 10 units of bovine intestinal alkaline phosphatase (Sigma–Aldrich, St. Louis, MO, P0114) at 37°C for 1 hr before Western blot analysis for phosphorylated RIP1 was performed as described above.