PIP3 is not required for RasC-mediated activation of the TORC2–PKBR1 pathway. (A) PHcrac-GFP translocation assay, which measures PIP3 content on the membrane, was performed in rasC⁻ cells induced to express Flag-RasC or -RasC^Q62L. (B) Phosphorylation of PKB was measured in wild-type (WT), pi3k1⁻/2⁻, and pi3k1⁻/2⁻ cells expressing Flag-RasC or -RasC^Q62L. The protein-transferred membrane was stained with Coomassie brilliant blue (CBB) and shown as loading control. (C) Phosphorylation of PKB was measured in wild-type and pten⁻ cells. The protein-transferred membrane was stained with Coomassie brilliant blue and shown as loading control. (B and C) Vertical black lines indicate that intervening lanes have been spliced out.