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. 2024 May 7;43(12):2424–2452. doi: 10.1038/s44318-024-00104-6

Figure 7. CENP-U competes with Wapl for binding to the Scc1-SA2 sub-complex.

Figure 7

(A) U2OS-LacO cells transiently expressing the indicated proteins and the mutants of EGFP-LacI-Scc1 (I337A/L341A) and Myc-SA2 (W334A) were stained with the antibody for the Myc-tag, and DAPI. Example images are shown. (B) U2OS-LacO cells transiently expressing the indicated proteins and the mutants of Myc-SA2 (F367A) and Myc-SA2 (F371A) were stained with the antibody for the Myc-tag and DAPI. Example images are shown. (C) Lysates prepared from HEK-293T cells transiently expressing Scc1-GFP and/or Myc-SA2 in the forms of WT and the indicated mutants were subjected to pull-down with GST or GST-CENP-U (1–60), followed by immunoblotting with antibodies for GFP and the Myc-tag, and CBB staining. (D) Lysates prepared from HEK-293T cells transiently co-expressing Scc1-GFP and Myc-SA2 (WT and the indicated mutants) were subjected to pull-down with GST or GST-CENP-U (1-60), followed by immunoblotting with antibodies for GFP and the Myc-tag, and CBB staining. Irrelevant lanes were removed. (E) Lysates prepared from HEK-293T cells transiently expressing Scc1-GFP and/or Myc-SA2 in the forms of WT and the indicated mutants were subjected to pull-down with MBP or MBP-Wapl (1–630), followed by immunoblotting with antibodies for GFP and the Myc-tag, and CBB staining. (F) Lysates prepared from HEK-293T cells transiently co-expressing Scc1-GFP and Myc-SA2 (WT and the indicated mutants) were subjected to pull-down with MBP or MBP-Wapl (1–630), followed by immunoblotting with antibodies for GFP and the Myc-tag, and CBB staining. (G) Lysates prepared from HEK-293T cells transiently expressing Scc1-GFP and Myc-SA2 were subjected to pull-down with GST-CENP-U (1–60) in the presence of increased amount of eluted MBP-Wapl (1–630) protein, followed by immunoblotting with antibodies for MBP, GFP and the Myc-tag, and CBB staining. (H) Lysates prepared from HEK-293T cells transiently expressing Scc1-GFP and Myc-SA2 were subjected to pull-down with GST-CENP-U (1–60) in the presence of increased amounts of eluted MBP-Wapl (1–630) protein (WT or the 3xEGE mutant), followed by immunoblotting with antibodies for MBP and the Myc-tag, and CBB staining. (I) Lysates prepared from HEK-293T cells transiently expressing Scc1-GFP and Myc-SA2 were subjected to pull-down with MBP-Wapl (1–630) in the presence of increased amounts of eluted GST-CENP-U (1–60) protein (WT or the ADA mutant), followed by immunoblotting with antibodies for GST, GFP, and the Myc-tag, and CBB staining. Data information: The white arrows point to the LacO repeats (A, B). Scale bars, 10 µm (A, B). Source data are available online for this figure.