Fig. 4.

Validation of the binding between AMPK complex and the candidate interaction proteins. A, Constructs encoding seven SFB-tagged AMPK subunits; the control genes LKB1, SIK1, or MFN1; or empty SFB construct were transfected into HEK293T cells. After 24 h, the cells were harvested, and the cell lysates were incubated with S-protein agarose and analyzed by Western blot analysis using the indicated antibodies. B, Constructs encoding the SFB-tagged AMPK complex binding candidates DCT, ENTR1, or Artemis; the control genes LKB1, SIK1, and MFN1; or empty SFB construct were transiently expressed in HEK293T cells. Cell lysates were subjected to pulldown assays with S-protein agarose and analyzed by Western blot analysis using the indicated antibodies. C, A TAP-MS analysis was performed using the candidate proteins interacting with the AMPK complex as the bait. The constructs encoding SFB-tagged Artemis, ENTR1, UBE2O, or DCT were transfected into HEK293T cells and selected with media containing 2 μg/ml puromycin. After TAP-MS, as shown in Fig. 1A, we identified the PSMs for each AMPK subunit. The number 1 or 2 on the x axis labels indicates two biological repeats for that gene.