Figure 2.

NOXA is ubiquitinated on lysine residues. (a) Cells were co-transfected with either NOXA-WT or NOXA-LL and pMT107, lysed in guanidium HCl and His-tagged proteins purified on Ni²⁺-NTA beads or lysed in RIPA buffer (WCL). (b) Cells were transfected as indicated, and Strep Tag-NOXA and associated proteins collected on streptactin beads. (c) NOXA-WT and -LL induce similar levels of apoptosis. HeLa cells were transfected with the indicated NOXA constructs or a control vector and apoptosis quantified after 48 h (top panel). Results are the mean ±S.E.M. (n=3). ^**P < 0.01 from control using a Student's t test. Proteins were assessed by western blotting (lower panel). (d) NOXA-WT and -LL, but not -3E interact with MCL-1. Cells were transfected with Strep Tag-NOXA-WT, -LL or -3E or a control vector. Strep Tag-NOXA and associated proteins were collected on streptactin beads. Asterisks denotes non-specific bands