Figure 3: MAGE-A4 promotes RAD18 stability.

(a) Recombinant RAD18–RAD6 complex (0, 0.27, 0.54 and 0.82 μM) was incubated with E1, ubiquitin and purified PCNA. Reaction products were analysed by immunoblotting with antibodies against the indicated proteins. (b) Soluble and chromatin fractions from H1299 cells were analysed by SDS–PAGE (20 μg per lane) and immunoblotting with antibodies against the indicated proteins. (c) H1299 cells were transiently transfected with an expression plasmid encoding CFP-RAD18 (or empty vector for control), ultraviolet irradiated (20 J m⁻²) and processed for immunofluorescence microscopy after 6 h. Scale bar, 10 μm. (d) H1299 and 293T cells were transfected with two independent siRNAs targeting MAGE-A4 or with control non-targeting siRNA oligonucleotides. After 72 h, extracts from the siRNA-transfected cells were analysed by immunoblotting with antibodies against the indicated proteins. (e,f) H1299 cells were transfected with siRNA oligonucleotides against MAGE-A4, RAD18 or control non-targeting siRNA as indicated. Forty eight hours later, cells were treated with cycloheximide (CHX, 100 μg ml⁻¹) and collected at different time points for immunoblot analysis.