Figure 2
PAR 2 -stimulated AMPK activation requires input from CAMKKβ and LKB-1. A. Representative western blot of pAMPK and tAMPK in lysates from NIH3T3 cells, pretreated with vehicle (control, left panel), with 10 μg/ml STO-609 (middle panel), or with 300nM BAPTA-AM (right panel), followed by treatment with 2fAP for 0-120 minutes. Bar graphs showing raw normalized pAMPK levels (B) or fold changes over baseline in normalized pAMPK (C) (n = 4). Baseline is defined as pAMPK observed in the absence of 2fAP in vehicle-treated cells. D-G. Cells were transfected with control (cnt) or LKB-1 siRNA and AMPK phosphorylation was determined as described above. Representative westerns showing anti-LKB-1 with and without siRNA knockdown (D) and phospho and total AMPK levels with and without LKB knockdown (E) are shown. Bar graphs show normalized pAMPK levels (F) or fold changes in pAMPK relative to baseline (G) after cnt or LKB-1 siRNA (n = 3). Baseline is defined as pAMPK observed in cntsiRNA transfected, untreated cells.