text: Luciferase Assay
  For Fig. 1, 10 ng of TORC1, 5 ng of TORC2, and 15 ng of PKAalpha expression plasmids were transfected into HEK293 cells. For Fig. 2, 800 ng of total DNA consisted of 200 ng of the pMLHoxb1ARE luciferase reporter and 100 ng of each expression plasmid. A lacZ reporter was co-transfected to normalize transfection efficiency. For Fig. 3A, the amounts of pENTR T2i shRNA (TORC2 shRNA) are given within the panel. For Fig. 3B, 25,000 cells per well were plated in 48-well plates and transfected using Lipofectamine 2000 with the following vectors: 40 ng of pML5xUAS, 40 ng of pGAL-DBD or pGAL-MEIS1A(335-390), 40 ng of pRSV-PBS or pRSV-PKA, 40 ng of pENTR U6 (control shRNA) or pENTR T2i shRNA (TORC2 shRNA), and 100 ng of pRSV-beta-galactosidase. At 48 h post-transfection, cell lysates were prepared using 100 mul per well of lysis buffer (1% Triton X-100, 1 mm dithiothreitol, 92.8 mm K2HPO4, pH 7.8, 9.2 mm KH2PO4, pH 7.8) and centrifuged for 5 min at 4 degreesC. A 20-mul aliquot of the supernatant was added to 12.5 mul of assay buffer (20 mm ATP, 40 mm MgCl2, 0.4 m Tris-Cl, pH 7.8). The mixture was immediately quantified for luciferase activity using a Lumat LB 9507 luminometer (EG&G Berthold) that dispensed 100 mul per reaction of luciferin solution, which contains 1 mm d-(-)-luciferin (catalogue number 11626353001, Roche) and 0.1 m Tris-Cl, pH 7.8.
  
entities: ['HEK293 cells']
entities: Cell
entities: PMC-2707216-sec-06_T1
entities: ['cells']
entities: Cell
entities: PMC-2707216-sec-06_T2
entities: ['cell lysates']
entities: Organism_substance
entities: PMC-2707216-sec-06_T3
entities: ['supernatant']
entities: Organism_substance
entities: PMC-2707216-sec-06_T4
entities: ['plasmids']
entities: Cellular_component
entities: PMC-2707216-sec-06_T5