Rescue of mTORC2-dependent TM phosphorylation protects Sin1^−/− Ab-MuLV pre-B cells from 17-AAG–mediated Akt degradation and cell death. (A) Total proteins from Sin1^−/− (knockout; KO), empty vector (KO + vector), or human Sin1 (KO + hSin1α)–reconstituted Sin1^−/− Ab-MuLV pre-B leukemia cells were assayed by immunoblotting for Akt and PKCβII TM phosphorylation as described in Figure 2A. ERK2 expression was used as a loading control. (B) Total cellular proteins were extracted from empty vector (+vector) or human Sin1 (+hSin1α)–reconstituted Sin1^−/− Ab-MuLV–transformed leukemia cells that were treated with 5μM 17-AAG for 0 or 8 hours. Total Akt protein levels were then measured by immunoblotting, with ERK2 serving as a loading control. The data shown are representative of 2 independent experiments. (C) Empty vector (vector) or hSin1α–reconstituted Sin1^−/− Ab-MuLV leukemia cells were treated with vehicle or 5μM 17-AAG for 24 hours. Cell viability was measured by PI and annexin V staining and flow cytometric analysis. A representative FACS plot is shown on the left. The numbers in the plot show the percentages of the gated populations in each quadrant. The graph on the right shows the relative proportion of live cells (PI⁻annexin V⁻) in each culture condition after 24 hours. The data shown are the average of triplicate samples with SD from 1 of 3 independent experiments. (D) The relative change in total cell number of vector or hSin1α–transduced Sin1^−/− Ab-MuLV pre-B cells cultured with or without 5μM 17-AAG for the indicated amount of time is shown. The data are the average of triplicate samples with SD from 1 of 3 independent experiments. The P values shown were calculated by a 2-tailed t test.