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Figure 4 | BMC Molecular Biology

Figure 4

From: Differential regulation of the two transcriptional activation domains of the coiled-coil coactivator CoCoA by sumoylation

Figure 4

Effect of sumoylation on the activity of CoCoA N-terminal AD. (A) 293T cells were transfected in 24-well plates with GK1-Luc reporter plasmid (150 ng) and plasmids encoding Gal4 DBD or Gal4 DBD-CoCoA fusion proteins (30, 60 or 100 ng) as indicated. Cell extracts were assayed for luciferase activity or analyzed by immunoblot with anti-Gal4 antibody 48 h after transfection. Luciferase activity results shown are from a single experiment which is representative of four independent experiments. (B) In vitro-translated HA-tagged CoCoA(1–190) wild type or mutant was incubated with GST, GST-p300 KIX fragment (left panel) or GST-β-catenin (right panel) fusion proteins bound to glutathione-Sepharose. Bound proteins were eluted and analyzed by immunoblot with anti-HA antibody (left panel) or autoradiography (right panel). (C) 293T cells were transfected in 24-well plates with pGL3OT reporter plasmid (100 ng), pSG5.HA-LEF1 (1 ng), pSG5.HA-β-catenin (1 ng), and a pSG5.HA vector encoding CoCoA(1–190), CoCoA(1–190, K29R), or SUMO1-fused CoCoA(1–190) (10–175 ng) as indicated. Cell extracts were assayed for luciferase activity or analyzed by immunoblot with anti-HA antibody 48 h after transfection. Luciferase activity results shown are from a single experiment which is representative of two independent experiments. RLU, Relative light units.

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