Figure 3.

MCL-1 stabilizes NOXA via a mechanism that requires NOXA–MCL-1 interactions. (a) MCL-1 selectively increases NOXA levels. Cells were co-transfected with plasmids encoding either Strep-tagged NOXA or a control and constructs encoding the indicated BCL-2 family members. A MyoD construct was also included as a control for equal transfection efficiency. Cell lysates were prepared and levels of the indicated proteins assessed by immunoblotting. (b) MCL-1 reduces NOXA turnover. Cells transfected with plasmids encoding Strep-tagged NOXA-WT in the presence or absence of MCL-1 were labeled with a ³⁵S-Cys/Met mixture for 1 h and chased with unlabeled Cys and Met. Strep-tagged proteins were captured on streptactin beads. Gels were subjected to autoradiography and ³⁵S-NOXA quantified using a phosphoimager. Results shown are from one of two experiments. (c) MCL-1 depletion lowers endogenous NOXA levels. HEK293T or HeLa cells were transfected with a pool of MCL-1 siRNAs and the indicated proteins assessed by immunoblotting. (d) MCL-1-dependent increases in NOXA levels require the NOXA BH3 domain. Cells were co-transfected with the indicated NOXA constructs or control vector and either a plasmid encoding MYC-MCL-1 or a control vector. After 24 h, lysates (WCL) were prepared and MYC-tagged MCL-1 and associated proteins precipitated with a MYC Tag mAb, and proteins assessed by immunoblotting