Figure 6
From: ERK2 phosphorylation of serine 77 regulates Bmf pro-apoptotic activity
Phosphorylation of serine 74 and serine 77 does not alter Bmf association with DLC2 and localization to the mitochondria. (a) HA-Bmf variants (DD, WT, S74A, S77A and AA) were co-expressed with FLAG-tagged DLC2 and Myc-tagged B-RAFV600E in 293FT cells for 24 h. Immunoprecipitation was performed using normal Rabbit IgG or antibody against the HA epitope tag. The presence of HA-Bmf and FLAG-DLC2 in immunoprecipitates and levels of transgene expression in cell lysates (input) was examined by western blot analysis using antibodies against HA and FLAG epitope tags. (b) WM793TR/HA-Bmf WT, S74A, S77A and AA cells were simultaneously treated with 100 ng/ml doxycycline (1 h) and 200 nM mitotracker Red CMXRos (30 min). Cells were then fixed for immunofluoresence analysis. HA-tag antibody was used to detect HA-Bmf (green, left panel). Mitochondria were highlighted by Red CMXRos dye (red, middle panel). The merged images of HA-Bmf and mitochondria staining are shown on the right. (c) Mitochondria fractionation was performed on doxycycline-treated WM793TR/HA-Bmf WT, S74A, S77A and AA cells. Expression of HA-Bmf in total lysates, mitochondria and cytosol fraction was detected by western blot. Actin and Cox IV were used as controls. (d) As in c except WM793TR/HA-Bmf WT DD cells were used
