Figure 2: MAGE-A4 associates with the RAD6-binding domain of RAD18.

(a) The indicated RAD18 fragments were expressed as GST fusions in E. coli. The RAD6-binding domain spanning residues 267–402 is highlighted in red. (b) GST–RAD18 fragments were incubated with H1299 cell extracts. After ‘pull-down’ with GSH-sepharose beads, the recovered GST–RAD18 fusions and 5% of ‘input’ H1299 cell lysate were analysed by immunoblotting with antibodies against GST, MAGE-A4 and RAD6. (c) GST–MAGE-A4 or GST was incubated with extracts from H1299 or 293T cells. After pulldown with GSH-sepharose beads, the recovered GST proteins (and 5% of input cell extract) were analysed by immunoblotting with antibodies against GST and RAD18. (d) Domain organization of full-length RAD18 and the RAD18 Δ340–395 (ΔR6BD) mutant harbouring an internal deletion that removes the RAD6-binding domain. (e) H1299 cells were transiently transfected with expression plasmids encoding HA–RAD18 and HA–RAD18 Δ340–395 (ΔR6BD) or with an empty vector control. Lysates from the resulting cells were immunoprecipitated with anti-HA antibodies. Anti-HA immune complexes and inputs (20 μg) were analysed by immunoblotting with antibodies against RAD18, MAGE-A4 and RAD6. (f) Recombinant GST, GST–RAD18 267–402 or GST–RAD6 were incubated with H1299 cell extracts then pulled down with GSH-sepharose beads. The recovered GST proteins were analysed by immunoblotting with antibodies against MAGE-A4 and GST. (g) Recombinant GST and GST–RAD18 267–402 were incubated with full-length recombinant Hexa-histidine-tagged MAGE-A4 (His-MAGE-A4). GST proteins were recovered using GSH-sepharose beads. Recovered GST proteins (and 5% of input) were analysed by immunoblotting with antibodies against GST and MAGE-A4.