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. 2022 Oct 5;12(10):220213. doi: 10.1098/rsob.220213

Figure 3.

Figure 3.

AMPK-dependent S314 phosphorylation activates NAMPT by facilitating NAMPT/PRPP association. (a) Bacterially purified unheated WT His/Flag-NAMPT or S314 mutant protein was incubated with purified active AMPK proteins for an in vitro kinase assay. His/Flag-NAMPT proteins were precipitated using anti-Flag antibody, and the NAMPT enzymatic activity in the precipitates was measured. ***p < 0.001. (b) HOK and HUVEC cells with expression of WT Flag-NAMPT or Flag-NAMPT S314A were treated with 10 Gy ionizing radiation. Flag-NAMPT proteins were precipitated from cell lysates 30 min after irradiation, washed twice with PBS, and the NAMPT enzymatic activity in the precipitates was measured. IR, ionizing radiation; WCL, whole cell lysate; **p < 0.01. (c) The NAMPT catalytic domain in human NAMPT protein (shown in cyan) structure was boxed by dotted line, and enlarged to show the spatial location of S314 (side chain shown in red), PRPP (oxygen atom shown in purple, PDB code: 2E5C), and nicotinamide (oxygen atom shown in purple, PDB code: 2E5D). (d) Human NAMPT protein structure (PDB code: 2E5C) shows the spatial location of S314 (side chain shown in red), PRPP (oxygen atom shown in purple), and H247 (side chain shown in orange). (e) Bacterially purified unheated WT His/Flag-NAMPT or S314 mutant protein was incubated with purified active AMPK proteins for an in vitro kinase assay. His/Flag-NAMPT proteins were precipitated using anti-Flag antibody, and the binding affinity between NAMPT protein and PRPP was measured. ***p < 0.001. (f) HOK cells with expression of WT Flag-NAMPT or Flag-NAMPT S314A were treated with 10 Gy ionizing radiation. Flag-NAMPT proteins were precipitated from cell lysates 30 min after irradiation, washed twice with PBS, and the binding affinity between NAMPT protein and PRPP was measured. **p < 0.01. (g) HOK cells with expression of WT Flag-NAMPT were pre-treated with 5 µM Compound C for 2 h, and cells were then treated with 10 Gy ionizing radiation. Flag-NAMPT proteins were precipitated from cell lysates 30 min after irradiation, washed twice with PBS and the binding affinity between NAMPT protein and PRPP was measured. **p < 0.01.