Shp2 mediates Mek/Erk1/2 pathway activation for survival downstream of the Kit mutant in leukemic cells. (A) Whole-cell extracts from 606HS2 and 931HS2 cells transduced with Shp2-shRNA77 or Shp2-shRNA78 or control ns-shRNA were analyzed by immunoblotting with the indicated Abs 72 hours after infection. (B) Lysates from 606HS2 and 931HS2 cells treated or not (−; DMSO) for 4 hours with UO126 (UO; 20μM) were analyzed by immunoblotting with the indicated Abs. In panels A and B, data are from 1 representative experiment of 5. (C) The amount of activated Ras (Ras-GTP) was measured by Ras-GTP pull-down assays using the Ras-GTP–binding domain of Raf1 and lysates from 606HS2 and 931HS2 cells treated with Kit inhibitors or not (−) and lysates from 606HS2 and 931HS2 cells transducing Shp2-shRNA77 or Shp2-shRNA78 or control ns-shRNA 72 hours after infection. Anti-Ras immunoblotting of the corresponding lysates was performed to control the amount of total Ras in each sample. Anti–β-actin Ab was used as a loading control. Data are from 1 representative experiment of 3. (D) Cells (606HS2 and 931HS2) were plated at 1 × 105 cells/mL and cultured for 48 hours in the absence (−; DMSO) or in the presence of the indicated concentrations of UO126. The number of living cells, the percentage of dead cells evaluated by trypan blue exclusion, and the percentage of apoptotic cells evaluated by flow cytometry using anti-active caspase 3 Abs were determined. Data are means ± SEM (n = 3). Statistical differences from the value of the control (−) are indicated as follows: **P < .01; ***P < .001 (by Student t test).
