Figure 2.

AMPK phosphorylates NAMPT S314 under ionizing radiation. (b–d, f–k) Immunoblotting analyses were performed using indicated antibodies. (a) HOK cells were pre-treated with 10 µM KU55933, 1 µM NU7441, 2 µM AZD6738, 20 µM PD98059, 20 µM SP600125 or 5 µM Compound C for 2 h, and cells were treated with 10 Gy ionizing radiation. Cellular NAMPT activity was measured 30 min after irradiation. **p < 0.01; ns, not significant. (b,c) HOK and HUVEC cells were treated with 10 Gy ionizing radiation, and cells were harvested 15 min after irradiation. Immunoblots (b) and immunoprecipitations (c) were performed using indicated antibodies. IR, ionizing radiation; WCL, whole cell lysate. (d) Bacterially purified His/Flag-NAMPT protein was incubated with purified active AMPK proteins (His-AMPKα1, untagged AMPKβ1 and untagged AMPKγ1) in the presence or absence of Compound C and [γ-³²P]-ATP for an in vitro kinase assay. Immunoprecipitation was performed using anti-Flag antibody, and radioactivity in the precipitates was measured by autoradiography. (e) Alignment analyses of NAMPT S314 or T304 was performed among indicated species. S314 and T304 were shown in red, and the residues matches AMPK phosphorylation consensus was shown in blue. (f) Bacterially purified WT His/Flag-NAMPT protein or indicated mutants were incubated with purified active AMPK proteins in the presence [γ-³²P]-ATP for an in vitro kinase assay. Immunoprecipitation was performed using anti-Flag antibody, and radioactivity in the precipitates was measured by autoradiography. (g) HOK cells were treated with 10 Gy ionizing radiation. Immunoblots were performed using indicated antibodies in the presence or absence or NAMPT pS314 blocking peptide. (h) HOK and HUVEC cells with expression of WT Flag-NAMPT or Flag-NAMPT S314A were treated with 10 Gy ionizing radiation, and immunoprecipitation was performed 30 min after irradiation. (i) HOK cells with expression of Flag-NAMPT were pre-treated with 5 µM Compound C for 2 h, and treated with 10 Gy ionizing radiation. Immunoprecipitation was performed 30 min after irradiation. (j) HOK cells were treated with 0.5 mM A769662 for 30 min. (k) HOK cells were incubated with glucose-free medium for 12 h. Glc, glucose.