17-AAG destabilizes Akt and PKCβII and induces cell death of Sin1^−/− Ab-MuLV pre-B leukemia cells preferentially. (A) Total cellular proteins from Sin1^+/− (wild-type; WT) or Sin1^−/− (knockout; KO) Ab-MuLV pre-B cells were assayed by immunoblotting for TM phosphorylation of Akt (p-T450) and PKCβII (p-T641). Sin1 expression is shown and ERK2 expression was used as a loading control. (B) Sin1 WT or KO Ab-MuLV pre-B cells were cultured in the presence or absence of 5μM 17-AAG for the indicated periods of time. Total Akt or PKCβII expression at each time point was measured by immunoblotting. ERK2 and β-actin expression served as loading controls. The Akt/actin or PKCβII/actin ratios were calculated by dividing the total pixel volume of Akt or PKCβII by the total pixel volume of β-actin. The results shown are representative of 2 independent experiments. (C) Sin1 WT or KO Ab-MuLV pre-B cells were cultured with or without 5μM 17-AAG for 24 hours. Cell viability was measured by flow cytometry with PI and annexin V staining. A representative FACS plot is shown on the left. The numbers in the plot show percentages of the gated populations in each quadrant. The data shown on the right graph are the average of triplicate samples from 1 of 3 independent experiments. (D) Sin1 WT or KO Ab-MuLV pre-B cells were cultured for 24 hours with or without 5μM 17-AAG and the relative change in viable cell number was determined. The data shown are the average of triplicate samples from 1 of 3 independent experiments. The P values shown were calculated by a 2-tailed t test.