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. 2010 Jul 26;190(2):233–245. doi: 10.1083/jcb.201001129

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© 2010 Cai et al.

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Figure 7. — RasC^Q62L activates TORC2-mediated phosphorylation of PKB in vitro. (A) Wild-type cell lysates were stimulated with GTP-γS alone or in the presence of GST or GST-RBD and then probed with phospho-specific antibodies. The protein-transferred membrane was stained with Coomassie brilliant blue (CBB) and shown as loading control. (B) The indicated cell lines were mixed, lysed, incubated in the presence or absence of GST or GST-RBD for 12 min, and then probed with phospho-specific antibodies. The protein-transferred membrane was stained with Coomassie brilliant blue and shown as loading control. Cells were not stimulated with chemoattractant, nor was GTP-γS added to the reaction. WT, wild type. (C) Quantitative densitometry of the time courses of reactions 3, 4, and 5 of B. The data represent mean ± SD of three independent experiments. A.U., arbitrary unit. (D) piaA⁻ cells expressing Flag-RasC^Q62L were mixed with wild-type cells in the presence of increasing concentrations of GDP-βS, filter lysed, incubated on ice for 12 min, and then probed with anti–phospho-HM antibody.