Reconstitution of PKB activation in vitro with immunopurified TORC2. (A) Cell lysates from piaA⁻ cells expressing Flag-RasC or -RasC^Q62L were mixed with HSS made from wild-type (WT) cells or piaA⁻ cells expressing Flag-PiaA or with Flag-eluted immunoprecipitate from Flag/piaA⁻ cells or Flag-PiaA/piaA⁻ cells. (B) Membrane fractions (MF) prepared from Flag-RasC/piaA⁻ or Flag-RasC^Q62L /piaA⁻ cells were mixed with Flag-eluted immunoprecipitate from Flag/piaA⁻ cells or Flag-PiaA/piaA⁻ cells. (C) Cell lysates from rip3⁻ cells expressing Flag-RasC or -RasC^Q62L were mixed with Flag-eluted immunoprecipitate from Flag/piaA⁻ cells or Flag-PiaA/piaA⁻ cells. (D) Cell lysates from piaA⁻ cells expressing Flag-RasC^Q62L were mixed with Flag-eluted immunoprecipitate from Flag-PiaA/piaA⁻ cells or Flag-PiaA/rip3⁻ cells. (E) Flag-eluted immunoprecipitate from Flag-PiaA/piaA⁻ cells was treated with increasing concentrations of PP242 before being mixed with cell lysates from piaA⁻ cells expressing Flag-RasC^Q62L. (F) Whole cell extracts were prepared from rip3⁻ cells expressing T7-Rip3 and Flag-RasC, T7-Rip3 and Flag-RasC^Q62L, or Flag-RasC^Q62L alone. Immunoprecipitation (IP) was performed using anti-T7 antibody, and samples were immunoblotted (IB) with anti-T7 or anti-Flag antibodies.