Figure 4: MAGE-A4 protects RAD18 from ubiquitin-mediated proteolysis.

(a) Replicate plates of H1299 cells were transfected with siRNA against MAGE-A4, USP7 or with non-targeting control siRNA. After 48 h, one plate of each replicate was treated with 10 μM MG132 for 16 h. Extracts from control and MG132-treated cells were analysed by immunoblotting with antibodies against the indicated proteins. (b) 293T cells were co-transfected with an HA–RAD18 expression vector in combination with a CMV-MAGE-A4 plasmid or an empty vector for control. After 48 h, RAD18 complexes were immunoprecipitated with anti-HA antibodies. The resulting immune complexes were incubated in a rabbit reticulocyte lysate (RRL) to reconstitute ubiquitin-coupled proteolysis in vitro. Relative levels of RAD18 and MAGE-A4 were determined by immunoblotting and quantified using densitometry. (c) H1299 cells were transiently co-transfected with WT or mutant HA–RAD18 expression plasmids in combination with a MAGE-A4 expression vector (or empty vector control). Forty eight hours later, cells were harvested for immunoblot analysis of RAD18 and MAGE-A4. The white arrowhead indicates the RAD18Δ340–395 mutant protein band that is insensitive to MAGE-A4. (d) Replicate cultures of H1299 cells were transfected with an expression vector encoding MYC–TRIM69 or with an empty vector plasmid for control. Sixteen hours later, the cells were transfected with siRNA against MAGE-A4 or with a scrambled control siRNA and incubated for an additional 48 h before immunoblot analysis.