Figure 2.

MAPK15 activates ULK1. A, HeLa cells were transfected with plasmid encoding for ATG13-FLAG in combination with HA-MAPK15^WT, HA-MAPK15^KD, ULK1^WT-MYC, ULK1^KD-MYC, or the empty vector. After 24 h, total lysates were analyzed by Western blot (WB) analysis (left panel). ATG13 was analyzed for phosphorylation levels (Ser³¹⁸), and scores were plotted on the graph (right panel, n = 3). B, HeLa cells were transfected with MAPK15 siRNA or SCR siRNA. After 48 h, cells were incubated in starvation medium (60 min), and total lysates were analyzed by Western blot analysis. Endogenous ULK1 was analyzed for phosphorylation (Ser³¹⁷) and total levels. Inhibition of Ser³¹⁷ phosphorylation of ULK1 (ratio P-ULK1 (Ser³¹⁷)/ULK1) induced by MAPK15 down-regulation by specific siRNAs has been expressed as a percentage of the siSCR-transfected sample (control, 100%). C, HeLa cells were transfected with plasmid encoding for HA-MAPK15 or the empty vector (−). After 24 h, cells were treated for 1 h with increasing concentrations of the SU6656 AMPK inhibitor, as indicated. Starved, positive control, cells were incubated in starvation medium for 60 min at 24 h after transfection with an empty vector. Total lysates were analyzed by Western blot analysis (left panel). Endogenous ULK1 was analyzed for phosphorylation (Ser³¹⁷) and total levels. HA-MAPK15 and MAPK1 levels were also analyzed by Western blotting for normalization purposes.