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. 2017 May 12;18(6):929–946. doi: 10.15252/embr.201744095

Figure 1. ZBTB48 binds telomeres via its ZnF11 domain in vitro and in vivo .

Figure 1

  1. Domain structure of wild‐type (WT) ZBTB48 containing a N‐terminal BTB domain and 11 zinc fingers (ZnF) at the C‐terminus of the 688‐amino‐acid protein. Note that ZnF2 is degenerate. Below is a schematic of the deletion construct lacking ZnF1‐10 used in (B).
  2. Sequence‐specific DNA pull‐downs with either telomeric (TTAGGG) or a control sequence (GTGAGT) for FLAG‐ZBTB48 WT, point mutants for the ten functional zinc fingers and a domain deletion construct for ZnF1‐10.
  3. Sequence‐specific DNA pull‐downs for FLAG‐ZBTB48 WT and ZnF11 point mutant for telomeric and subtelomeric variant repeat sequences (green) and their respective scrambled controls (blue).
  4. Co‐localization analysis of endogenous ZBTB48 and TRF2 in U2OS cells by immunofluorescence (IF) staining. A representative image illustrating the co‐localization between ZBTB48 (green) and TRF2 (red) as a marker for telomeres is shown with DAPI (blue) used as a nuclear counterstain. The quantification of frequency of co‐localization events (right) was done after 3D reconstruction of the acquired z‐stacks (n = 104 cells). The average value is indicated by a red bar.
  5. IF stainings for exogenous FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11 in U2OS cells. The same analysis as in (D) was performed and average co‐localization frequencies are shown (n = 24–33 cells). Error bars indicate standard deviations, and P‐values are based on Student's t‐test.
  6. Co‐localization analysis between ZBTB48 (green) and PML (red) as a marker for PML bodies analogous to (D) in U2OS cells (n = 102 cells).
Data information: (D–F) Co‐localization events are indicated by white arrows. Scale bars represent 5 μm.Source data are available online for this figure.