Decreased aldolase activity in AMPK KO T cells. WT vs AMPK KO T cells were transplanted into B6D2F1 recipients, recovered on day 7, and proteins immunoprecipitated from cell lysates using an antibody detecting the phosphorylated AMPK-specific motif LxRxx(pS/pT). Precipitated candidate proteins were subsequently identified via LC-MS and a heat map generated of those recovered at threefold or higher levels in WT vs AMPK KO T cells (A). Representative LC-MS data from the heat map in panel A is shown for the 4 glycolytic enzymes of interest (B). Control protein samples were recovered from day 7 samples before immunoprecipitation and blotted for total levels of candidate proteins aldolase and GAPDH (C). (D-E) WT or AMPK KO T cells were stimulated on CD3/CD28 coated plates for 72 hours, followed by T-cell recovery and measurement of aldolase activity in cell lysates. GAPDH was measured in a similar fashion from T cells recovered on day 7 posttransplant (F). For data in panels A-B, 12× WT and 12× AMPK KO T cells were recovered on day 7 after transplant and divided into 4 groups of 3 recipients each. These 4 groups were then processed as individual replicates through cell lysis, IP, and LC-MS analysis. In panels C,F, n = 3 replicates pooled from ≥9 individual recipients (eg, 3 groups of 3 recipients each). Graphs in panels D-E represent data from 3 separate biological donors. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. PKM, pyruvate kinase M.