Figure 2. Okadaic acid inhibits autophagy.

(a) Okadaic acid (OA) inhibits the dephosphorylation of ULK1 at S637 but not S757. MEF, U2OS or HT1080 cells were incubated in starvation media or starvation media with 200 nM OA for 1 h. Phosphorylation of S637 and S757 on ULK1 was monitored using site specific phospho-antibodies. (b) MEFs were treated as in a and lysates were probed for three reported mTOR substrates (S6K, 4EBP1 and DAP1) and endogenous LC3. (c) OA inhibits starvation-induced autophagy. MEFs stably expressing GFP-LC3, GFP-ULK1 or GFP-ATG16 were incubated in starvation media or starvation media containing 200 nM OA for 1 h. Cells were fixed and imaged for GFP-LC3, GFP-ULK1 or GFP-ATG16 translocation. Representative images from two independent experiments shown. Average number of puncta per cell were quantitated and expressed as such or as fold change relative to full media (fold change or mean±s.d., n=20. two-tail Student's t-test, *P<0.05).