Figure 5

C-Raf kinase does not phosphorylate VDAC protein. A. In vitro kinase assay was performed using GST-C-Raf recombinant protein and HA-VDAC immunoprecipitated from 293 cells. HA-VDAC was incubated with recombinant active or inactive GST-C-Raf proteins in presence of radioactive ATP. As a positive control GST-C-Raf was incubated with MEK kinase dead substrate in the same conditions. The blots were also probed with anti-HA and anti-C-Raf antibodies. B. In vivo phospholabelling was performed by coexpressing HA-VDAC with different C-Raf constructs in 293 cells. Transfected cells were incubated with radioactive orthophosphate before HA-VDAC immunoprecipitation. HA-Bad was used as a positive control.