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. 2019 Feb 21;104(8):1597–1607. doi: 10.3324/haematol.2018.192773

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Figure 3. — Identification of KLF4 as a novel binding partner of RUNX1 and RUNX1-ETO. (A) KLF4 co-localized with RUNX1 in nuclei. (Top) Co-localization analysis of exogenous MYC-RUNX1 and FLAG-KLF4 in 293T cells at 48 h after transfection by immunofluorescence assay. Anti-MYC (green) and anti-FLAG (red) antibodies were used as primary antibodies; DAPI (blue) was used for nuclear staining. Scale bar represents 10 μm. (Middle and bottom) Co-localization analysis of endogenous RUNX1 and KLF4 in Kasumi-1 and SKNO-1 cells with anti-RUNX1 (green) and anti-KLF4 (red) antibodies; DAPI (blue) was used for nuclear staining. Scale bar represents 10 μm. (B) KLF4 co-localized with RUNX1-ETO in nuclei. (Top) Co-localization analysis of exogenous MYC-RUNX1-ETO and FLAG-KLF4 in 293T cells at 48 h after transfection. Antibodies were used as described in (A). Scale bar represents 10 μm. (Middle and bottom) Co-localization analysis of endogenous RUNX1-ETO and KLF4 in Kasumi-1 and SKNO-1 cells with anti-ETO (green) and anti-KLF4 (red) antibodies. DAPI (blue) was used for nuclear staining. Scale bar represents 10 μm. (C) KLF4 interacted with RUNX1. 293T cells were co-transfected with pCMV5-MYC-RUNX1 and pCMV5-FLAG-KLF4. At 48 h after transfection cells were harvested and cell lysates underwent immunoprecipitation (IP) with anti-FLAG or anti-MYC antibody. Immunoblotting (IB) analysis was performed with the other antibody. (D) KLF4 interacted with RUNX1-ETO. 293T cells were co-transfected with pCMV5-MYC-RUNX1-ETO and pCMV5-FLAG-KLF4. At 48 h after transfection, cells were harvested and cell lysates underwent IP with anti-FLAG or anti-MYC antibody. Immunoblotting analysis was performed with the other antibody. WB: western blotting.