FIGURE 11.

Interaction domains in PR-B and MAGE-11. A, pCMV-FLAG empty vector (−) (5 μg) and 5 μg of pCMV-FLAG-PR-B-(1–164) wild type (WT) and L110A,L111A, V114A,L115A, and L118A,L119A mutants were expressed in COS cells with 4 μg of pSG5-HA-MAGE. Cells were incubated with 1 μm MG132 for 24 h before harvest. Immunoprecipitates (IP) and cell extracts (30 μg of protein/lane) were probed on the transblot using HA and FLAG antibodies. B, GALO empty vector (−) or GAL-PR-B-(1–164) WT or L110A,L111A, V114A,L115A, and L118A,L119A mutants (50 ng) were expressed in HeLa cells with 0.1 μg of 5XGAL4Luc3 and 0.1 μg of pSG5 empty vector (−), pSG5-MAGE (M), or pSG5-HA-p300 (P). Luciferase activity (mean ± S.E.) is representative of three independent experiments. C, top, schematic diagram of full-length human MAGE-11 with Lys-240 (K240) and Lys-245 (K245) monoubiquitinylation (Ub) sites and cell cycle checkpoint kinase Chk1 phosphorylation site Thr-360 (T360) in F-box residues 329–369 in the MAGE homology domain (MHD). Bottom, GAL-PR-B-(1–164) (50 ng) was expressed in HeLa cells with 0.1 μg of 5XGAL4Luc3 and 0.1 μg of VP-MAGE WT or K240A,K245A, T360A, V333A,M334A, or L358A,L359A mutants. Inset, VP16 empty vector (−); VP-MAGE WT; and K240A,K245A, T360A, V333A,M334A, and L358A,L359A mutants (10 μg) were expressed in COS cells. Cells were incubated with 1 μm MG132 prior to harvest. The transblot of cell extracts (60 μg of protein/lane) was probed using VP16 antibody. Error bars, S.E.