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. 2009 Oct 14;284(50):34793–34808. doi: 10.1074/jbc.M109.065979

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© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 8. — MAGE-11 and TIF2 interaction domains. A, schematic diagram of full-length MAGE-11 and deletion fragments, with nuclear localization signal (NLS, residues 18–22), monoubiquitinylation sites Lys-240 and -245, Ser-174 and Thr-360 phosphorylation sites (19), MAGE homology domain (MHD), and the F-box region. B, FLAG-b empty vector (—) (8 μg) (lanes 1–5) or 8 μg of FLAG-TIF2 (lanes 6–10) were expressed in COS cells with 0.5 μg of pSG5-HA-MAGE-(112–429), 1 μg of pSG5-HA-MAGE-(112–307), or 2 μg of pSG5-HA-MAGE-(165–307), -(112–298), and –(112–276). Cells were incubated with 0.1 μg/ml of EGF and 1 μm MG132, solubilized in IP lysis buffer and immunoprecipitated using FLAG affinity resin overnight at 4 °C. IP (upper panel) and cell extracts (lower panels) (15 μg of protein/lane for HA-MAGE, 55 μg of protein/lane for FLAG-TIF2) were analyzed on immunoblots probed using HA and FLAG antibodies. C, FLAG-b empty vector (lanes 1 and 2) (—), FLAG-TIF2 (lanes 3 and 4), FLAG-TIF2.0-(1–627), or FLAG-TIF2.8-(1011–1179) (8 μg) were expressed in COS cells with 5 μg of pSG5-HA-MAGE or 0.5 μg of pSG5-HA-MAGE-(112–429). Cells were incubated with 0.1 μg/ml of EGF and 1 μm MG132, extracted in IP lysis buffer, and incubated with FLAG affinity resin overnight at 4 °C. Immunoprecipitates (upper panel) and cell extracts (lower panels) (25 μg of protein/lane) were analyzed on immunoblots probed using FLAG and HA antibodies. D, FLAG-b empty vector (—) (8 μg) (lanes 1–3) and FLAG-TIF2.0-(1–627) (lanes 4–6) were expressed in COS cells with 1 μg of pSG5-HA-MAGE-(112–429), 2 μg of pSG5-HA-MAGE-(165–429), or 2 μg of pSG5-HA-MAGE-(112–307). Cells were incubated with 0.1 μg/ml of EGF and 1 μm MG132, extracted in IP lysis buffer, and incubated with FLAG resin for 1 h at 4 °C. Immunoprecipitated proteins (upper panel) and cell extracts (lower panels) (25 μg of protein/lane) were analyzed on immunoblots probed using HA and FLAG antibodies. E, HeLa cells were transfected with 0.05 μg of GAL-MAGE-(85–205) wild-type (WT), S174A or S174D mutant, 0.1 μg of 5×GAL4Luc3, and 0.05 μg of VP16 empty vector (—) or VP-TIF2.0-(1–627). Bottom panel, GAL0 empty vector (—) (2 μg) and 2 μg of GAL-MAGE-(85–205) WT, S174A, or S174D mutant were expressed in HeLa cells. Cell extracts (200 μg of protein/lane) were analyzed on immunoblots probed using a GAL antibody.