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. 2013 May 21;288(27):19459–19470. doi: 10.1074/jbc.M113.475087

FIGURE 5.

FIGURE 5.

Expression of CBL mutants results in constitutive phosphorylation of S6 and enhanced survival of TF-1 cells. A, SHP2 immunoprecipitations (IP) were performed on lysates collected from TF-1 cells expressing CBL-WT, CBL-Y371H, and CBL-C384R, which were depleted of cytokines overnight and stimulated with GM-CSF. B, lysates collected were also probed with phospho-Ser-325/326 S6 and total S6. C, TF-1 cells expressing wild type CBL, CBL-Y371H, and CBL-C384R were depleted of cytokine overnight and stimulated with GM-CSF. Lysates collected were probed with phospho-STAT (pSTAT5) or phospho-ERK (pErk) and reprobed for total STAT5 or ERK. D, TF-1 cells expressing WT-CBL or CBL mutants Y371H or C384R were depleted of cytokine overnight. Starved cells were plated in 96-well plates and incubated for 48 h, prior to the addition of XTT reagent. Reduction of XTT reagent was measured using a spectrometer (n = 4). **, p ≤ 0.001; ***, p ≤ 0.0001. E, TF-1 cells expressing wild type or CBL mutants were maintained in cytokine-free media for 72 h. The cells were then stained with annexin V and analyzed via flow cytometry (n = 3; *, p ≤ 0.05). Error bars, S.E.