Figure 5

Analysis of the regulation of anti-apoptotic and pro-apoptotic proteins in JAK2 ^V617F mutant SET-2 cells. A: JAK2 wild type TF-1 cells (left panels) were starved overnight in medium containing 0.1% FCS without GM-CSF. Cells were then stimulated with 2 ng/ml GM-CSF (+GM) for 8 hours and extracted. Control TF-1 cell extracts (Ctrl) were prepared from cells growing in full medium. SET-2 cells (right panels) were treated with DMSO (Ctrl) or 500 nM NVP-BSK805 for 16 hours. Cells were either extracted or washed and released into fresh medium (FM) without NVP-BSK805 for 8 hours and then extracted. B: SET-2 cells were treated with DMSO or 500 nM NVP-BSK805 for 4 or 24 hours, extracted, and lysates were subjected to co-immunoprecipitation studies. To control for potential unspecific binding of the proteins of interest to beads, extracts (same total protein input as used for the immunoprecipitations) were also incubated with beads without the respective antibodies (Ctrl). C: Levels of soluble Bak, Bax, Mcl-1, Bcl-xL and Bim in SET-2 whole cell extracts following treatment of cells for 4 and 24 hours with DMSO or NVP-BSK805. D: SET-2 cell lysates from cells treated as described above in panel B were subjected to immunoprecipitation of Bax (using an antibody recognizing an amino-terminal epitope) and co-immunoprecipitation studies. Potential unspecific binding of the proteins of interest to beads was controlled (Ctrl) as described above. E: SET-2 cells were incubated with DMSO for 48 hours or with 500 nM NVP-BSK805 for the indicated times. Bak activation was assessed using a Bak active conformation-specific antibody by flow cytometry analysis. Results are depicted as the means of three independent experiments ± SD. *Significantly different from DMSO control using t-test (p < 0.05). **Significantly different from DMSO control using t-test (p < 0.001).