Fig. 4: G9a-mediated methylation inhibits interactions between RUNX3 and CBFβ/p300.
From: RUNX3 methylation drives hypoxia-induced cell proliferation and antiapoptosis in early tumorigenesis
A Co-immunoprecipitation (IP) assay of CBFβ and RUNX3 in HEK293 cells transfected with control vector and G9a plasmids (WT, ∆SET). Immunoprecipitates were analyzed by immunoblotting with anti-CBFβ, anti-RUNX3, anti-G9a, and anti-β-actin antibodies. B Co-immunoprecipitation (IP) assay of p300 and RUNX3 in HEK293 cells transfected with control vector, p300 (HA-p300) and G9a plasmids (WT, ∆SET). Immunoprecipitates were analyzed by immunoblotting with anti-acetyl Lysine (#9441, Cell Signaling Technology), anti-RUNX3, anti-p300 (ab10485, abcam), anti-G9a, and anti-β-actin antibodies. C Co-immunoprecipitation (IP) assay of CBFβ and RUNX3 in HEK293 cells transfected with G9a plasmid (pEGFP-G9a) and indicated RUNX3 mutants. Immunoprecipitates were analyzed by immunoblotting with anti-CBFβ, anti-RUNX3, anti-G9a, and anti-β-actin antibodies. D Co-immunoprecipitation (IP) assay of p300 and RUNX3 in HEK293 cells transfected with G9a plasmid (pEGFP-G9a) and indicated RUNX3 mutants. Immunoprecipitates were analyzed by immunoblotting with anti-RUNX3, anti-p300, anti-acetyl Lysine, anti-G9a, and anti-β-actin antibodies. Protein levels were quantified from the western blot images (n = 3), and data are shown as the mean ± SD. *p < 0.05 (CBFβ and RUNX3) and #p < 0.05 (Ace-RUNX3) by one-way ANOVA with Tukey’s posthoc correction. E Co-immunoprecipitation (IP) assay of RUNX3 and importins in HEK293 cells transfected with RUNX3 (Flag-RUNX3), importins (Myc-importin-α1, Myc-importin-α7), and/or CBFβ (HA-CBFβ) plasmids. Immunoprecipitates were analyzed by immunoblotting with anti-Myc (ab9106, abcam), anti-HA (SC-7392, Santa Cruz), anti-Flag (F1804, Sigma), anti-CBFβ (ab124693, abcam), anti-RUNX3, and anti-Tubulin (SC-5286, Santa Cruz) antibodies. F Co-immunoprecipitation (IP) assay of RUNX3 and importin-α1 in HEK293 cells transfected with RUNX3 (Flag-RUNX3), importins (Myc-importin-α1), and/or G9a (pEGFP-G9a) plasmids. Immunoprecipitates were analyzed by immunoblotting with anti-Myc, anti-HA, anti-GFP, anti-RUNX3, anti-importin-α1, anti-G9a, and anti-β-actin antibodies. G Co-immunoprecipitation (IP) assay of RUNX3 and importin-α7 in HEK293 cells transfected with RUNX3 (Flag-RUNX3), CBFβ (HA-CBFβ), importin (Myc-importin-α7) and/or G9a (pEGFP-G9a) plasmids. Immunoprecipitates were analyzed by immunoblotting with anti-Myc, anti-HA, anti-Flag, anti-GFP, anti-RUNX3, anti-CBFβ, anti-importin-α7, anti-G9a, and anti-β-actin antibodies.
