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. 2015 Nov 5;290(52):30975–30987. doi: 10.1074/jbc.M115.669531

FIGURE 2.

FIGURE 2.

Fbw7 through its WD domain physically associates with Runx2 in its Runx2ΔNΔC region. a, Runx2 and its deletion mutants containing different regions with exact amino acids are schematically represented for both mouse Runx2 (mRunx2, isoform 5, 1–528 amino acids) and human Runx2 (hRunx2, isoform 2, 1–507 amino acids). b, Lysates of 293T cells transfected with 0.5 μg of Runx2 or Runx2ΔNΔC either alone or together with Fbw7α and its deletion mutants (Fbw7αΔF, Fbw7αWD) were immunoblotted (IB) with anti-FLAG-M2 and anti-Runx2 antibodies. c, lysates of 293T cells transfected with 0.5 μg of human Runx2 or its deletion mutants (all contained two of the potential CPDs present in Runx2ΔNΔC) either alone or together with Fbw7α were immunoblotted with anti-FLAG-M2 and anti-Runx2 antibodies. d, Fbw7 co-immunoprecipitates (IP) using anti-Fbw7 antibody from lysates of MC3T3-E1 cells were immunoblotted with anti-Fbw7 and anti-Runx2 antibodies. Cells were treated with MG132 (10 μm) 6 h before lysate preparation. e, Fbw7 co-immunoprecipitates using anti-FLAG-M2 antibody from lysates of MC3T3-E1 cells transfected with 0.5 μg of Fbw7 were immunoblotted with anti-FLAG-M2 and anti-Runx2 antibodies. Cells were treated with MG132 (10 μm) 6 h before lysate preparation and lithium chloride (20 mm) 24 h before lysate preparation. f, Fbw7 co-immunoprecipitates from lysates of MC3T3-E1 cells transfected with 50 nm siGSK3β were immunoblotted with anti-Fbw7, anti-GSK3β, and anti-Runx2 antibodies. g, Fbw7 co-immunoprecipitates using anti-FLAG M2 antibody from lysates of 293T cells transfected with 1.0 μg of Runx2 or its deletion mutants (Runx2ΔNΔC, Runx2C, or Runx2N) either alone or together with FLAG-Fbw7α were immunoblotted with anti-FLAG-M2 and anti-Runx2 antibodies. h and i, Fbw7 co-immunoprecipitates using anti-Fbw7 antibody from lysates of HEK293T cells transfected with 1.0 μg of FLAG-Runx2N or FLAG-Runx2C either alone or together with FLAG-Fbw7 were immunoblotted with anti-FLAG-M2 antibody. IgG-HC: heavy chain; IgG-LC: light chain. j, Fbw7 co-immunoprecipitates using anti-FLAG M2 antibody from lysates of MC3T3-E1 cells transfected with 0.5 μg of FLAG-Fbw7α or its deletion mutants (Fbw7αΔF or Fbw7αWD) were immunoblotted with anti-FLAG-M2 and anti-Runx2 antibodies. Data are representative of minimum three independent experiments.