Figure 5: Mutational analyses to define structural requirements for MAGE-A4-induced RAD18 stabilization.

(a) Domain structure of full-length MAGE-A4 and MAGE-A4 mutants used in this study. The MAGE-homology domain (MHD) is conserved between MAGE family members and comprises juxtaposed WH-A and WH-B regions. (b) 293T cells were transiently transfected with expression vectors encoding the MAGE-A4 mutants shown in a or with an empty vector (EV). After 48 h, extracts from the resulting cells were analysed by immunoblotting with anti-Pan-MAGE-A (which recognizes an epitope in the WH-B domain) or with anti-MAGE-A4 (which recognizes a C-terminal epitope of MAGE-A4 in residues 275–317). (c) Replicate plates of 293T cells were transiently transfected with expression vectors encoding WT or mutant forms of MAGE-A4. Forty-eight hours post transfection, cells were treated with cycloheximide (CHX) and then harvested at different times post CHX. Cell extracts were analysed by immunoblotting with antibodies against RAD18, MAGE-A4 and actin. (d) RAD18 levels in each lane of immunoblots in c were quantified by densitometry with ImageJ software. The graph indicates the levels of RAD18 remaining at each time point following CHX treatment in control and MAGE-A4-expressing cells.