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. 1998 May;18(5):2867–2875. doi: 10.1128/mcb.18.5.2867

FIG. 6.

FIG. 6

Complex formation of GSK-3β, Axil, and β-catenin. (A) Complex formation in intact cells. The lysates (20 μg of protein) of COS cells expressing HA–GSK-3β and Myc-Axil (lanes 1 and 3) and HA–GSK-3β alone (lanes 2 and 4) were probed with the anti-Myc, anti-β-catenin, and anti-HA antibodies (lanes 1 and 2). The same lysates (500 μg of protein) were immunoprecipitated with the anti-HA antibody, and the immunoprecipitates were probed with the anti-Myc, anti-β-catenin, and anti-HA antibodies (lanes 3 and 4). IP, immunoprecipitation, Ab, antibody; Ig, immunoglobulin. The arrow, large arrowhead, and small arrowhead indicate the positions of Myc-Axil, endogenous β-catenin, and HA–GSK-3β, respectively. (B) Deletion mutants of Axil. The hatched and empty boxes indicate the RGS and Dsh homologous domains, respectively. (C) Expression of Axil deletion mutants and their interaction with GSK-3β and β-catenin. The lysates (20 μg of protein) of COS cells expressing Myc-Axil(full length) (lane 1), Myc-Axil(1-670) (lane 2), Myc-Axil(682-838) (lane 3), Myc-Axil(1-265) (lane 4), and Myc-Axil(265-483) (lane 5) were probed with the anti-Myc antibody (left panel). The same lysates (500 to 1,000 μg of protein) (lanes 6 to 10) were immunoprecipitated with the anti-Myc antibody, and the immunoprecipitates were probed with the anti-GSK-3β and anti-β-catenin antibodies (right panel). The small and large arrowheads indicate the positions of endogenous GSK-3β and β-catenin, respectively. (D) Different binding sites of Axil for GSK-3β and β-catenin. After GST–GSK-3β (lanes 1 to 4) and GST–β-catenin (lanes 5 to 8) (8 pmol each) were incubated with MBP-Axil(265-483) (lanes 1 and 5), MBP-Axil(265-412) (lanes 2 and 6), MBP-Axil(412-483) (lanes 3 and 7), or MBP (lanes 4 and 8) (2 pmol each) immobilized on amylose resin, MBPs fused to proteins were precipitated by centrifugation. The precipitates were probed with the anti-GSK-3β and anti-β-catenin antibodies. The small and large arrowheads indicate the positions of GST–GSK-3β and GST–β-catenin, respectively. (E) Phosphorylation of Axil(265-483) by GSK-3β. MBP-Axil(265-483) (200 ng of protein) was incubated with (lane 2) or without (lane 1) GST–GSK-3β (100 ng of protein) for 30 min. The arrow indicates the position of MBP-Axil(265-483). The results shown are representative of three independent experiments.