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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 11. — MAGE-11 FXXIF motif-dependent interaction with TIF2. A, top panels, FLAG-b empty vector (lane 1) (—) or FLAG-TIF2.8-(1011–1179) (lanes 2–4) (8 μg) were expressed in COS cells with 0.5 μg of pSG5-HA-MAGE-(112–429) wild-type (WT) (lanes 1 and 2) or the indicated mutant (lanes 3 and 4). Cells were incubated with 0.1 μg/ml of EGF and 1 μm MG132 and immunoprecipitated overnight at 4 °C using FLAG resin. IP (upper panel) and cell extracts (25 μg of protein for HA-MAGE, 50 μg of protein for FLAG-TIF2.8, lower panels) were analyzed on immunoblots using HA and FLAG antibodies. Bottom panels, HeLa cells were transfected with 2 μg of pSG5 (—), pSG5-HA-MAGE-(2–429) WT, F260A, or F264A mutant (left panel), and pSG5-HA-MAGE-(112–429) WT, F260A, or F264A mutant, and pSG5 (—) (right panel). Cells extracts in IB lysis buffer (150 μg of protein/lane) were analyzed on immunoblots using HA antibody. B, GAL-TIF2.1-(624–1287) or GAL-TIF2.3m123 (TIF2-(624–1179)-L644E,L645A,L693A,L694A,L748A,L749A) (0.05 μg) were transfected in HeLa cells with 0.1 μg of pSG5 (—), pSG5-HA-MAGE-(112–429) WT or mutant, and 0.1 μg of 5×GAL4Luc3. C, CV1 cells were transfected with 0.1 μg of pCMV-AR and 5 μg of PSA-Enh-Luc with and without 2 μg of pSG5-TIF2 and 1 μg of WT or mutant pSG5-HA-MAGE. Cells were incubated in the absence and presence of 1 nm DHT. Bottom panel, CV1 cells were transfected with 8 μg of pSG5 (–), pSG5-HA-MAGE-(112–429) WT, F260A, or F264A mutant. Cells extracts in IB lysis buffer (200 μg of protein/lane) were analyzed on immunoblots probed with HA antibody. D, CV1 cells were transfected with 0.1 μg of pCMV-ARΔ120–472 with and without 2 μg of pSG5-TIF2, 2 μg of WT or mutant pSG5-HA-MAGE, and 5 μg of MMTV-Luc. Cells were incubated in the absence and presence of 1 nm DHT. Bottom panel, CV1 cells were transfected with 8 μg of pSG5 (—), pSG5-HA-MAGE WT, F151A, F260A, or F264A mutant. Cell extracts in IB lysis buffer (150 μg/lane) were analyzed on immunoblots using HA antibody. E, HeLa cells were transfected with 0.1 μg of 5×GAL4Luc3, 50 ng of GAL0 empty vector (—), WT or mutant GAL-MAGE-(251–272), and 0.1 μg of VP16 empty vector (—), VP-TIF2.0-(1–627), VP-TIF2.1-(624–1287), or VP-TIF2.2-(1288–1464). Bottom panel, HeLa cells were transfected with 2 μg of GAL0 (lane 1), GAL-MAGE-(251–272) WT (lane 2), or F260A,F264A mutant (lane 3). Cell extracts (100 μg of protein/lane) were analyzed on immunoblots using GAL antibody.