Figure 3. TORC1 antagonizes Nem1 phosphorylation.

(A, B) SDS-PAGE (A) and Phos-tag phosphate-affinity gel electrophoresis (B) analyses of plasmid-encoded Nem1-HA₃ in exponentially growing nem1Δ cells treated with rapamycin (RAP) for the indicated times. The levels of Pgk1 served as loading controls in (A). (C) Phosphorylation pattern analysis of Nem1-PtA on Phos-tag gels. Plasmid-encoded Nem1-PtA was purified from exponentially growing (EXP) and rapamycin-treated (RAP; 30 min) nem1Δ cells and treated with (+), or without (−), alkaline phosphatase (AP) in the absence (−), or presence (+), of phosphatase inhibitors (PPI). P0, P1, and P2 (in B and C) denote 3 differentially phosphorylated Nem1-HA₃ or Nem1-PtA isoforms.